Inhibitors of platelet activation and recruitment

ABSTRACT

The present invention provides soluble CD39 polypeptides and compositions, and methods for inhibiting platelet activation and recruitment in a mammal comprising administering a soluble CD39 polypeptide.

REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of pendingInternational Application No. PCT/US99/22955, filed Oct. 13, 1999, whichwas published under PCT Article 21 (2) on April 27 2000, in English, asWO 00/23459, and which claims the benefit of U.S. ProvisionalApplication Serial No. 60/104,585, filed Oct. 16, 1998, now abandoned,and U.S. Provisional Application Serial No. 60/107,466, filed Nov. 6,1998, now abandoned, and U.S. Provisional Application Serial No.60/149,010, filed Aug. 13, 1999, now abandoned. InternationalApplication No. PCT/US99/22955 and U.S. Provisional Application SerialNos. 60/104,585, 60/107,466, and 60/149,010 are incorporated herein byreference.

FIELD OF THE INVENTION

[0002] This invention relates to soluble CD39 compounds andcompositions, the preparation thereof, and the use thereof to inhibitplatelet activation and recruitment in a mammal.

BACKGROUND OF THE INVENTION

[0003] CD39 is a cell-surface antigen that was originally identified asa marker for mature B cells, but is also expressed on less mature Bcells, Epstein-Barr Virus-transformed B cells, activated T cells,endothelial cells and some myeloid cell lines (Dörken et al., inLeukocyte Typing IV; W. Knapp, B. Dörken, and W. R. Gilks, Eds; OxfordUniversity Press, New York, N.Y.; pp. 89-90, 1989). Monoclonalantibodies against CD39 induce B cell homotypic adhesion, an activitythat may be important in the regulation of immune function (Kansas andTedder, J. Immunol. 147:4094-4102, 1991). Molecular cloning andcharacterization of CD39 indicated that it is unique cell surfacemolecule that contains two potential transmembrane regions and ahydrophobic segment within the putative extracellular domain(Maliszewski et al., J. Immunol. 153:3574, 1994). The amino acidsequence of CD39 was reported to exhibit some homology with a guanosinediphosphatase from yeast (Maliszewski et al., supra).

[0004] In 1996, an ATP diphosphohydrolase was cloned from potato tubers(Handa and Guidotti, Biochem. Biophys. Res. Commun. 218:916, 1996). Theamino acid sequences of this and several other NTPases demonstrated ahigh degree of similarity, particularly within several small “apyraseconserved regions” (ACR). CD39 shares these conserved regions withsoluble ATP-diphosphorylase from potato tubers, other apyrases andrelated enzymes. It was subsequently reported that native andrecombinant full-length CD39 possess E-type ATP diphosphohydrolase(ATPDase) activity (Marcus et al., J. Clin. Invest. 99:1351, 1997);Kaczmarek et al., J. Biol. Chem. 271:33116, 1996); Wang and Guidotti, J.Biol. Chem. 271:9898, 1996). ATPDases degrade nucleoside tri- and/ordiphosphates, but not monophosphates (Plesner, Int. Rev. Cytol. 158:141,1995).

[0005] Vascular endothelial cells constituitively express a cell-surfaceADPase (ecto-ATP diphosphohydrolase, apyrase, EC 3.6.1.5), one of atleast 3 thromboregulatory systems which function in the maintenance ofblood fluidity (Marcus and Safier, FASEB J. 7:516, 1983; Marcus et al.,J. Clin. Invest. 88:1690, 1991). This ecto-ADPase, which belongs to theE-type ATPDase family, rapidly metabolizes ADP in the plateletreleasate, terminating further platelet recruitment and aggregation.

[0006] Immunoprecipitation of HUVEC detergent lysates with anti-CD39 mAbresulted in complete capture of cell-associated ADPase activity,suggesting that CD39 is the only ecto-ADPase on endothelial cells(Marcus et al., J. Clin. Invest. 99:1351, 1997). In the same study, COScell transfectants expressing recombinant CD39 at the cell surfacetotally inhibited ADP-induced platelet aggregation. Thus, CD39 plays aprominent role in thromboregulation (see also, Gayle et al., J. Clin.Invest., 101:1851, 1998).

[0007] Excessive platelet activation (i.e., stimulation by an agonist)and recruitment, leading to platelet aggregation and vessel occlusion atsites of vascular injury in the coronary, carotid, and peripheralarteries, presents a major therapeutic challenge in cardiovascularmedicine. Excessive platelet activation and recruitment is acontributing factor in clinical disorders including stroke, unstableangina, myocardial infarction, and restenosis following percutaneouscoronary intervention including angioplasty, atherectomy, stentplacement, and bypass surgery.

[0008] Glycoprotein IIb/IIIa antagonists, such as the monoclonalantibody marketed as ReoPro® (Centocor Inc.), are presently underdevelopment for the inhibition of platelet aggregation in patientsundergoing percutaneous coronary intervention, and in patients withacute coronary syndromes such as unstable angina and myocardialinfarction. The activation of glycoprotein IIb/IIIa receptors, however,is a late event in the cascade that leads to platelet aggregation.

[0009] There is a great need to identify additional therapeuticstrategies and compositions for the pharmacological neutralization ofplatelet reactivity (activation, recruitment, aggregation). Inparticular, there is a need to identify compounds and compositions whichtarget early portions of coagulation pathways such as the ADP-dependentactivation and recruitment of platelets. There is, in fact, an urgentneed to identify new strategies and compositions for the treatment ofstroke, which is the third leading cause of death in the United States.In the case of stroke, an advantageous therapeutic agent will reduceintravascular thrombus burden and accompanying neurological defectswithout increasing intracerebral hemorrhage.

SUMMARY OF THE INVENTION

[0010] Soluble forms of CD39 having apyrase activity constitute a novelapproach to the prevention and/or treatment of disease. The presentinvention provides soluble CD39 polypeptides and nucleic acids,compositions comprising a pharmaceutically acceptable carrier and asoluble CD39 polypeptide, and methods of making and using soluble CD39polypeptides having apyrase activity. The effectiveness of soluble CD39polypeptides have been demonstrated in vitro, ex vivo, and in vivo.

[0011] The invention is directed to soluble CD39 polypeptides selectedfrom the group consisting of:

[0012] (a) polypeptides having an amino acid sequence as set forth inFIG. 1 (SEQ ID NO:2) wherein the amino terminus is selected from thegroup consisting of amino acids 36-44, and the carboxy terminus isselected from the group consisting of amino acids 471-478; (b) fragmentsof the polypeptides of (a) wherein said fragments have apyrase activity;(c) variants of the polypeptides of (a) or (b), wherein said variantshave apyrase activity; and (d) fusion polypeptides comprising thepolypeptides of (a), (b), or (c), wherein said fusion polypeptides haveapyrase activity. The invention provides compositions comprising apharmaceutically acceptable carrier and a soluble CD39 polypeptide.

[0013] The invention is also directed to nucleic acids encoding asoluble CD39 polypeptide. The invention provides DNAs, vectors,recombinant cells, and recombinant methods for the production of solubleCD39 polypeptides.

[0014] The invention is further directed to the use of soluble CD39polypeptides for inhibiting platelet activation and recruitment, forinhibiting angiogenesis, or for degrading nucleoside tri- and/ordi-phosphates in a mammal in need of such treatment. The inventionencompasses the use of a soluble CD39 polypeptide for the preparation ofa medicament for inhibiting platelet activation and plateletrecruitment, for inhibiting angiogenesis, or for degrading nucleosidetri- and/or di- phosphates in a mammal in need of such treatment. Theseand other aspects of the present invention will become evident uponreference to the following drawings, examples, and detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015]FIG. 1 shows the predicted amino acid sequence (SEQ ID NO:2) ofhuman CD39. The predicted amino acid sequence contains 6 potentialN-linked glycosylation sites (double underline), and 11 cysteineresidues (bold face). The two predicted transmembrane regions areunderlined (single underline).

[0016]FIG. 2 shows the domain structure of full length CD39 and of anengineered soluble form of CD39. The locations of transmembrane regionsnear the amino- and carboxy-termini, the centrally located hydrophobicsequence, and a section containing the four putative apyrase conservedregions (ACR) are indicated. Cysteine residues are marked as “C”. Thesoluble CD39 contains a FLAG® peptide and new leader sequence and lacksthe two transmembrane regions.

[0017]FIG. 3 shows the immunoaffinity depletion of solCD39 from COS-1conditioned medium (CM) following one (1X) or two (2X) rounds ofadsorption. Samples were assayed for ATPase activity as described inExample 7. Data are expressed as pmoles of ATP degraded per minute.

[0018]FIG. 4 shows the immunoprecipitation of solCD39 from COS-1 CM.Lane 2 shows the material that specifically bound to the antibody-coatedbeads. Lane 1 shows material that was pre-incubated withovalbumin-coated beads to remove non-specifically bound material priorto addition of Ab-coated beads. Migration of molecular weight standardsis indicated in kilodaltons (kDa).

[0019]FIG. 5 shows the immunoaffinity purification and characterizationof soluble CD39 (solCD39). FIG. 5A shows fractions from theimmunoaffinity column analyzed by SDS-PAGE, FIG. 5B shows enzymeactivity in the fractions, and FIG. 5C shows purified solCD39 before(Lane 1) and after (Lane 2) treatment with N-glycanase.

[0020]FIG. 6A shows pH optimum profiles of HUVEC membrane ecto-ADPase(•) and recombinant solCD39 (▪). FIG. 6B shows is an Eadie-Hofstee plotof rates of metabolism at different concentrations of ATP (•) or ADP (▪)using purified solCD39 (6.5 ng).

[0021]FIG. 7 shows inhibition of ADP-induced platelet reactivity bypurified solCD39 in platelet-rich plasma from a donor who had ingestedaspirin. The response to increasing concentrations of ADP is shown inFIG. 7A. The effect of increasing quantities of purified solCD39 on theplatelet aggregation response to 10 μM ADP is shown in FIG. 7B. Arrowsindicate the addition of agonist. Data are presented as relative lighttransmission vs. time (4 min duration).

[0022]FIG. 8 shows a comparison of platelet reactivity as modulated bydifferent agonists and inhibitors. The effects of CM from cellsexpressing solCD39 on platelet aggregation induced by 5 μM ADP (FIG. 8A)and collagen (FIG. 8B) were compared in PRP and PRP treated with 10 μMindomethacin. In FIG. 8B, 1 μg/ml collagen was used in the upper samplesand 3.3 μg/ml in the lower (indomethacin-treated) samples. FIG. 8C showsthe inhibition of collagen-induced aggregation by increasing quantitiesof solCD39 in PRP from a donor who had ingested aspirin. The arrowsindicate the addition of agonist. Data are presented as relative lighttransmission vs time (4 min.).

[0023]FIG. 9 shows the effect of FSBA-treated solCD39 on plateletreactivity. FIG. 9A shows the effects of purified solCD39, FSBA-treatedsolCD39, and mock-treated solCD39 (each at 4.4 μg/ml) on ASA-treated PRPafter addition of 10 μM ADP. FIG. 9B shows the effects of FSBA-treatedsolCD39 and mock-treated solCD39 (each at 22 μg/ml) on ASA-treated PRPfollowing addition of 3.3 μg/ml collagen. FIG. 9C shows the titration ofmock-treated solCD39 (0.88-2.2 μg/ml) against FSBA-treated solCD39 (22μg/ml). ASA-treated PRP was stimulated with 10 μM ADP. Arrows indicateaddition of agonist. Data are presented as relative light transmissionvs time.

[0024]FIG. 10 shows pharmacokinetic analyses of solCD39 in mice. CD39 inserum was measured in the radioactive phosphate release ATPase assay (▪)or the ADPase assay (•). Activities are expressed as pmoles nucleotidedegraded per minute. The dashed line indicates the ATPase activity of 25μg/ml of solCD39 in murine serum. Distribution (t_(½)α=59 min (ATP); 43min (ADP)) and clearance (t_(½)=40 h (ATP & ADP)) half-lives weredetermined using a biphasic curve fit.

[0025]FIG. 11 shows bleeding times at 0 and 60 minutes in pigs treatedwith low, medium, or high doses of solCD39.

[0026]FIG. 12 shows the effect of aspirin on pig platelet aggregation atbaseline and day 5 after intravenous administration (FIG. 12A) and theeffect of effect of high dose solCD39 on platelet aggregation atbaseline and day 7 (FIG. 12B).

[0027]FIG. 13 shows the inhibition of pig platelet aggregation by low,medium, and high doses of solCD39 as a function of time after bolusadministration.

[0028]FIG. 14 shows the concentration of CD39 in pig serum as a functionof time after low, medium, or high dose administration. Distribution(t_(½)α=29 min) and clearance (t_(½)β=51 h) half-lives were determinedusing a biphasic curve fit.

[0029]FIG. 15 shows the ex vivo aggregation of murine platelets.Platelets were stimulated with 10 μM ADP (FIG. 15A), 2.5 μg/ml collagen(FIG. 15B), or 0.1 mM sodium arachidonate (FIG. 15C) after theadministration of vehicle (saline), soluble CD39 (4 mg/kg) or aspirin (5mg/kg). Soluble CD39 treatment produced aggregation curves that returnedto baseline following stimulation with agonists, but aspirin treatmentyielded such a pattern only when arachidonate was the agonist.

[0030]FIG. 16 shows reversal of the ADP-induced aggregation response inmurine platelets when solCD39 is added at the peak of the aggregationresponse.

[0031]FIG. 17 shows the inhibition of platelet (n=20, FIG. 17A) andfibrin (n=3, FIG. 17B) deposition following induction of stroke in micepretreated with 8 mg/kg soluble CD39. “Fibrin” is a positive control,“Ipsilat” is ipsilateral (i.e., the ischemic hemisphere), and“Contralat” is the nonischemic hemisphere.

[0032]FIG. 18 shows the comparative effects of vehicle (n=23), solubleCD39 (n=67) and aspirin (n=27) on the outcome of induced stroke in mice.FIG. 18A shows cerebral blood flow, 18B shows cerebral infarct volume,18C shows neurological score (where higher scores indicate a worsedeficit (Connolly, E. S., Jr., et al., Neurosurg. 38(3):523-532 (1996)),18D shows mortality, and 18E shows intracerebral hemorrhage. *p<0.05,^(‡)p<0.01, ^(†)p<0.001.

[0033]FIG. 19 shows a covariate plot of cerebral infarct volume vs.intracerebral hemorrhage. Vehicle (saline), aspirin (ASA, 5 mg/kg priorto stroke), soluble CD39 (4 & 8 mg/kg, prior to stroke), and solubleCD39 (8 mg/kg, 3 h following stroke induction in mice) are compared.

[0034]FIG. 20A shows the construct used to generate CD39-/- mice byhomologous recombination. The labeled restriction sites are BglII (B),Spel (S), and Asp718 (A). FIG. 20B shows a genomic Southern blot as usedto identify ES clones having a disrupted CD39 allele.

[0035]FIG. 21 shows the bleeding times in control (n=15),aspirin-treated (5 mg/kg, n=10), solCD39-treated (4, 8, and 20 mg/kg,n=25) and solCD39-/- mice (n=10). (*p<0.05, ^(‡)p<0.01, ^(†)p<0.001).

[0036]FIG. 22 shows a comparison of stroke outcomes in control(C57BL/6J×129/J F1) mice (n=6), CD39-/- mice (n=5), and CD39/- micewhich were “reconstituted” with solCD39 (n=6). FIG. 22A shows cerebralblood flow, 22B shows cerebral infarct volume, 22C shows neurologicalscore, 22D shows mortality, and 22E shows intracerebral hemorrhage.*p<0.05, ^(‡)p<0.01, ^(†)p<0.001.

[0037]FIG. 23 is a Kaplan-Meier plot showing that solCD39 causes animprovement in survival in a stringent lung ischemia-reperfusion model.

[0038]FIG. 24 shows an alignment of the N-terminal amino acid sequencesof human CD39 and human CD39-L4.

DETAILED DESCRIPTION OF THE INVENTION

[0039] A cDNA encoding the cell-surface molecule CD39 has been isolated,cloned and sequenced. The nucleic acid sequence and predicted amino acidsequence of this cDNA are shown in SEQ ID NO:1 and SEQ ID NO:2. Thepresent invention provides methods of using soluble forms of CD39, whichwere constructed by removing the amino- and carboxy-terminaltransmembrane domains. Soluble CD39 retains the capacity of wildtypeCD39 to metabolize ATP and ADP at physiologically relevantconcentrations as well as the ability to block and reverse ADP-inducedplatelet activation and recruitment, including platelet aggregation. Theuse of soluble forms of CD39 is advantageous because purification of thepolypeptides from recombinant host cells is facilitated, and becausesoluble polypeptides are generally more suitable than membrane-boundforms for clinical administration. Because CD39 inhibits plateletactivation and recruitment, and therefore platelet aggregation, thepresent invention provides methods and compositions for inhibitingformation of a thrombus at a site in a mammal at which platelets areinappropriately activated, methods for use in controlling plateletreactivity, thereby regulating the hemostatic and thrombotic processes,and methods of inhibiting and/or reversing platelet aggregation.

[0040] A. Hemostasis

[0041] Hemostasis is defined as the arrest of bleeding from damagedblood vessels, and results from a sequence of physiologic andbiochemical events. At least three interacting biological systems areinvolved in hemostasis: components of the blood vessels (such as thesubendothelial matrix), platelets, and plasma proteins (Marcus, A. J.:Disorders of Hemostasis, Ratnoff and Forbes, eds., W. B. Saunders,Philadelphia, 1996; pages 79-137; Marcus, A. J.: Platelet Activation,in: Atherosclerosis and Coronary Artery Disease, vol.1, Fuster, Ross andTopol, eds., Lipincott-Raven, Philadelphia, 1996; pages 607-637). Adefect or defects in one or more of these systems can result inhemorrhagic disorder; conversely, the inappropriate activation ofhemostasis culminates in development of arterial or venous thrombosis.

[0042] When a blood vessel is injured, it contracts, exposingsubendothelial matrix components such as collagen, von Willebrandfactor, fibronectin, thrombospondin, laminin, and microfibrils.Platelets adhere to, and are activated by, these components; collagen isan especially effective agonist for platelet activation. At least fourphysiologic events are initiated by platelet-collagen contact: theplatelets release biologically active compounds; they express P-selectinon their cell surface (where it mediates adhesion of neutrophils,monocytes and subsets of lymphocytes); the platelet eicosanoid pathwayis activated (starting with the liberation of arachidonic acid whichforms prostaglandin H₂); and the platelets undergo a drastic change inshape, from smooth disks to spiny spheres.

[0043] The biologically active compounds released by platelets arenumerous, and multi-functional. Included in this group of components areserotonin, ATP, ADP, calcium, adhesive proteins (fibrinogen,fibronectin, thrombospondin, vitronectin, von Willebrand factor), growthfactors (platelet-derived growth factor, transforming growth factor-β,platelet factor 4) and coagulation factors (factor V, high-molecularweight kininogen, factor XI, protein S and plasminogen activatorinhibitor-I (PAI-I)). Some of these compounds play a role in therecruitment of additional platelets and/or other cells such asneutrophils and monocytes to the site of activation, whereas others areinvolved in feedback mechanisms to down-regulate excessive thrombusformation.

[0044] At least three separate endothelial thromboregulatory systemsexist: the eicosanoids including the prostaglandins PGI₁ and PGD₂;endothelium-dependent relaxing factor (EDRF/NO); and theecto-nucleotidase ATP-diphosphohydrolase (ATPDase) which has both ADPaseand ATPase activities. While collagen and thrombin are the primeinducers of platelet secretion, ADP is the most important agonist ofplatelet aggregation present in the platelet releasate. Catabolism ofADP to AMP by the ecto-ADPase blocks further recruitment of additionalplatelets to the site, reverses the aggregation response and blockssubsequent thrombus response.

[0045] Ecto-nucleotidase activity is demonstrable in vitro in anaggregrometry system in which EDRF/NO effects and PGI₂ production areblocked by hemoglobin and aspirin respectively (Marcus and Safier, FASEBJ 7:516; 1993). In this system, loss of platelet stimulatory activity inthe supernatant fluid correlates with ADP catabolism. An ADPase activityhas been identified in the membrane fraction of human endothelial cells;enzyme activity detected by polyacrylamide gel electrophoresis indicatedboth ATPase and ADPase (Marcus et al., Clin. Res. 40:226A (abstract),1992).

[0046] B. Utility of the Claimed Invention

[0047] Significant research efforts are directed to the discovery andcharacterization of platelet aggregation inhibitors because of thepotential utility of such inhibitors in treating occlusive vasculardisease. For example, WO 95/12412 discloses platelet-specific chimericantibodies and methods of using the same in treating various thromboticdisorders. A prototype description of the efforts to develop thistherapeutic agent and obtain approval for its use as a human therapeuticagent (generic name: abciximab, trade name: ReoPro®) was described by B.S. Coller in Circulation 92:2373 (1995).

[0048] CD39 is an ecto-ADPase (apyrase) located on the surface ofendothelial cells. This enzyme is mainly responsible for the maintenanceof blood fluidity, thus maintaining platelets in the baseline (resting)state. This is accomplished by metabolism of the major platelet agonist,adenosine diphosphate, to adenosine monophosphate, which is not anagonist. Because ADP is the most important agonist of plateletaggregation, and is present in platelet releasate, a substance whichcatabolizes ADP is useful in treating or preventing disease states thatinvolve inappropriate aggregation of platelets.

[0049] Examples of the therapeutic uses of soluble CD39 and compositionsthereof include the treatment of individuals who suffer from coronaryartery disease or injury following myocardial infarction,atherosclerosis, arteriosclerosis, preeclampsia, embolism,platelet-associated ischemic disorders including lung ischemia, coronaryischemia, and cerebral ischemia, and for the prevention of reocclusionfollowing thrombosis, thrombotic disorders including coronary arterythrombosis, cerebral artery thrombosis, intracardiac thrombosis,peripheral artery thrombosis, venous thrombosis, thromboticmicroangiopathies including thrombotic thrombocytopenic purpura (TTP)and hemolytic uremic syndrome (HUS), essential thrombocythemia,disseminated intravascular coagulation (DIC), and thrombosis andcoagulopathies associated with exposure to a foreign or injured tissuesurface, in combination with angioplasty, carotid endarterectomy,anastomosis of vascular grafts, and chronic cardiovascular devices suchas in-dwelling catheters or shunts. Other instances in which it would beuseful to inhibit increased ADP release due to increased plateletstimulation would be in individuals at high risk for thrombus formationor reformation (severe arteriosclerosis), and inhibition of occlusion,reocclusion, stenosis and/or restenosis of blood vessels. Individualswho will benefit from therapies that involve inhibiting ADP-inducedaggregation of platelets include those at risk for advanced coronaryartery disease, and those that are or will be undergoing angioplastyprocedures (i.e., balloon angioplasty, laser angioplasty, coronaryatherectomy and similar techniques), placement of endovascularprosthetic devices such as carotid, coronary, peripheral arterial orother endovascular stents, dialysis access devices, or procedures totreat peripheral vascular disease. Inhibition of platelet aggregationwill also be useful in individuals undergoing surgery that has a highrisk of thrombus formation (i.e., coronary bypass surgery, insertion ofa prosthetic valve or vessel and the like), and in the prevention ortreatment of postpump syndrome (including the cerebral function declineattributed to microvascular thrombosis in patients followingcardiopulmonary bypass procedures), deep venous thrombosis (DVT),pulmonary embolism (PE), transient ischemic attacks (TIAs) and otherrelated conditions where arterial occlusion or microvascular thrombosisare common underlying features. In addition, the ability of CD39 toblock platelet activation and recruitment is useful for preventingstroke and for treating patients experiencing stroke due to vascularocclusion. In particular, the methods, compounds, and compositions ofthe present invention have the ability to inhibit microvascularthrombosis, improve postischemic cerebral blood flow, and reducecerebral infarction volumes and neurological deficit without inducingintracerebral hemorrhage, in stroke. Soluble CD39 and compositionsthereof according to the present invention can also be administered inany other therapeutic setting where it would be useful to degradenucleoside tri- and/or diphosphates. As an example, soluble CD39 may beused during the treatment of sepsis, and/or the treatment of variousinfections caused by bacterial pathogens such as Pseudomonas (includingthe organisms that colonize the lungs of cystic fibrosis patients inbiofilm-like structures despite aggressive antibiotic treatment),Klebsiella, Vibrio, Salmonella, Escherichia, Mycobacterium, Neisseria,and Helicobacter species. As a further example, soluble CD39 may be usedas an anti-neoplastic agent to inhibit angiogenesis and/or prevent thesurvival benefits that ATP provides to tumor cells, or to treat otherdiseases or conditions mediated by angiogenesis such as occularneovascularization.

[0050] Soluble CD39 polypeptides also have many non-therapeutic uses,since they may be used in any application where soluble ATPase and/orADPase activity is advantageous. As an example, soluble CD39polypeptides may be used in compositions for preserving platelets suchas those described by Gepner-Puszkin (U.S. Pat. No. 5,378,601). Asanother example, soluble CD39 polypeptides may be used inpyrophosphate-based DNA sequencing methodologies such as those describedby Ronaghi et al. (Science 281:336, 1998). As a further example, solubleCD39 polypeptides can be used to screen for apyrase inhibitors.

[0051] C. CD39 Polypeptides

[0052] The molecular cloning and structural characterization of CD39 ispresented in Maliszewski et al. (J. Immunol. 153:3574, 1994). CD39contains two putative transmembrane regions, near the amino and carboxytermini, which may serve to anchor the native protein in the cellmembrane. The portion of the molecule between the transmembrane regionsis external to the cell. As used herein, the term “CD39 polypeptides”includes CD39, homologs of CD39, variants, fragments, and derivatives ofCD39, fusion polypeptides comprising CD39, and soluble forms of CD39polypeptides.

[0053] Soluble polypeptides are polypeptides that are capable of beingsecreted from the cells in which they are expressed. A secreted solublepolypeptide may be identified (and distinguished from its non-solublemembrane-bound counterparts) by separating intact cells which expressthe desired polypeptide from the culture medium, e.g., bycentrifugation, and assaying the medium (supernatant) for the presenceof the desired polypeptide. The presence of the desired polypeptide inthe medium indicates that the polypeptide was secreted from the cellsand thus is a soluble form of the polypeptide. The use of soluble formsof CD39 is advantageous for many applications. Purification of thepolypeptides from recombinant host cells is facilitated, since thesoluble polypeptides are secreted from the cells. Moreover, solublepolypeptides are generally more suitable than membrane-bound forms forparenteral administration and for many enzymatic procedures.

[0054] Apyrase activity resides in the extracellular domain of CD39.Thus, for applications requiring biological activity, useful CD39polypeptides include soluble forms of CD39 such as those having an aminoterminus selected from the group consisting of amino acids 36-44 of SEQID NO:2, and a carboxy terminus selected from the group consisting ofamino acids 471-478 of SEQ ID NO:2, and which exhibit CD39 biologicalactivity. Soluble CD39 polypeptides also include those polypeptideswhich include part of either or both of the transmembrane regions,provided that the soluble CD39 polypeptide is capable of being secretedfrom a cell, and retains CD39 biological activity. Soluble CD39polypeptides further include oligomers or fusion polypeptides comprisingthe extracellular portion of CD39, and fragments of any of thesepolypeptides that have biological activity.

[0055] The term “biological activity,” as used herein, includes apyraseenzymatic activity as well as the ex vivo and in vivo activities ofCD39. Apyrases catalyze the hydrolysis of nucleoside tri- and/or di-phosphates, but a given apyrase may display different relativespecificities for either nucleoside triphosphates or nucleosidediphosphates. Biological activity of soluble forms of CD39 may bedetermined, for example, in an ectonucleotidase or apyrase assay (e.g.ATPase or ADPase assays), or in an assay that measures inhibition ofplatelet aggregation. Exemplary assays are disclosed herein; those ofskill in the art will appreciate that other, similar types of assays canbe used to measure biological activity.

[0056] Among the soluble CD39 polypeptides provided herein are variants(also referred to as analogs) of native CD39 polypeptides that retain abiological activity of CD39. Such variants include polypeptides that aresubstantially homologous to native CD39, but which have an amino acidsequence different from that of a native CD39 because of one or moredeletions, insertions or substitutions. Particular embodiments include,but are not limited to, CD39 polypeptides that comprise from one to tendeletions, insertions or substitutions of amino acid residues, whencompared to a native CD39 sequence. The CD39-encoding DNAs of thepresent invention include variants that differ from a native CD39 DNAsequence because of one or more deletions, insertions or substitutions,but that encode a biologically active polypeptide. Included as variantsof CD39 polypeptides are those variants that are naturally occurring,such as allelic forms and alternatively spliced forms, as well asvariants that have been constructed by modifying the amino acid sequenceof a CD39 polypeptide or the nucleotide sequence of a nucleic acidencoding a CD39 polypeptide.

[0057] Generally, substitutions for one or more amino acids present inthe native polypeptide should be made conservatively. Examples ofconservative substitutions include substitution of amino acids outsideof the active domain(s), and substitution of amino acids that do notalter the secondary and/or tertiary structure of CD39. Additionalexamples include substituting one aliphatic residue for another, such asIle, Val, Leu, or Ala for one another, or substitutions of one polarresidue for another, such as between Lys and Arg; Glu and Asp; or Glnand Asn. Other such conservative substitutions, for example,substitutions of entire regions having similar hydrophobicitycharacteristics, are known in the art.

[0058] When a deletion or insertion strategy is adopted, the potentialeffect of the deletion or insertion on biological activity must beconsidered. Subunits of the inventive polypeptides may be constructed bydeleting terminal or internal residues or sequences. Additional guidanceas to the types of mutations that can be made is provided by acomparison of the sequence of CD39 to polypeptides that have similarstructures, as well as by performing structural analysis of theinventive polypeptides.

[0059] The native sequence of full length CD39 is set forth in FIG. 1(SEQ ID NO:2). In some preferred embodiments the CD39 variants are atleast about 70% identical in amino acid sequence to the amino acidsequence of native CD39 as set forth in the sequence listing; in somepreferred embodiments the CD39 variants are at least about 80% identicalin amino acid sequence to the amino acid sequence of native CD39 as setforth in the sequence listing. In some more preferred embodiments thevariants of CD39 are at least about 90% identical in amino acid sequenceto the amino acid sequence of native CD39 as set forth in the sequencelisting; in some more preferred embodiments the variants of CD39 are atleast about 95% identical in amino acid sequence to the amino acidsequence of native CD39 as set forth in the sequence listing. In somemost preferred embodiments, variants of CD39 are at least about 98%identical in amino acid sequence to the amino acid sequence of nativeCD39 as set forth in the sequence listing; in some most preferredembodiments, variants of CD39 are at least about 99% identical in aminoacid sequence to the amino acid sequence of native CD39 as set forth inthe sequence listing. Percent identity, in the case of both polypeptidesand nucleic acids, may be determined by visual inspection. Percentidentity may be determined using the alignment method of Needleman andWunsch (J. Mol. Biol. 48:443, 1970) as revised by Smith and Waterman(Adv. Appl. Math 2:482, 1981. Preferably, percent identity is determinedby using a computer program, for example, the GAP computer programversion 10.x available from the Genetics Computer Group (GCG; Madison,Wis., see also Devereux et al., Nucl. Acids Res. 12:387, 1984). Thepreferred default parameters for the GAP program include: (1) a unarycomparison matrix (containing a value of 1 for identities and 0 fornon-identities) for nucleotides, and the weighted comparison matrix ofGribskov and Burgess, Nucl. Acids Res. 14:6745, 1986, as described bySchwartz and Dayhoff, eds., Atlas of Protein Sequence and Structure,National Biomedical Research Foundation, pp. 353-358, 1979 for aminoacids; (2) a penalty of 30 (amino acids) or 50 (nucleotides) for eachgap and an additional 1 (amino acids) or 3 (nucleotides) penalty foreach symbol in each gap; (3) no penalty for end gaps; and (4) no maximumpenalty for long gaps. Other programs used by one skilled in the art ofsequence comparison may also be used. For fragments of CD39, the percentidentity is calculated based on that portion of CD39 that is present inthe fragment.

[0060] The primary amino acid structure of soluble CD39 may be modifiedto create CD39 derivatives by forming covalent or aggregative conjugateswith other chemical moieties, such as glycosyl groups, lipids,phosphate, acetyl groups and the like. Covalent derivatives of CD39 areprepared by linking particular functional groups to CD39 amino acid sidechains or at the N-terminus or C-terminus of a CD39 polypeptide or theextracellular domain thereof. CD39 derivatives also include CD39polypeptides bound to various insoluble substrates, including cyanogenbromide-activated agarose structures, or similar agarose structures, oradsorbed to polyolefin surfaces (with or without glutaraldehydecross-linking).

[0061] Fusion polypeptides of soluble CD39 within the scope of thisinvention include covalent or aggregative conjugates of CD39 or itsfragments with other polypeptides, such as by synthesis in recombinantculture as N-terminal or C-terminal fusions. One class of fusionpolypeptides are discussed below in connection with soluble CD39oligomers. As another example, a fusion polypeptide may comprise asignal peptide (which is also variously referred to as a signalsequence, signal, leader peptide, leader sequence, or leader) at theN-terminal region or C-terminal region of a CD39 polypeptide whichco-translationally or post-translationally directs transfer of thepolypeptide from its site of synthesis to a site inside or outside ofthe cell membrane or cell wall (e.g. the α-factor leader ofSaccharomyces; several leader sequences are discussed in the examplesthat follow). It is particularly advantageous to fuse a signal peptidethat promotes extracellular secretion to the N-terminus of a solubleCD39 polypeptide. In this case, the signal peptide is typically cleavedupon secretion of the soluble CD39 from the cell.

[0062] In a particularly preferred embodiment, one or more amino acidsare added to the N-terminus of a soluble CD39 polypeptide in order toimprove the expression levels and/or stability of the CD39 polypeptide.The one or more amino acids include an Ala residue, fragments derivedfrom the N-terminus of another member of the CD39 family (e.g., CD39L2,CD39L3, CD39L4) or from another polypeptide such as IL-2, and otherpeptides, either naturally-occurring or designed based upon structuralpredictions, capable of adopting a stable secondary structure.

[0063] In a most preferred embodiment, a soluble CD39 polypeptide isinitially synthesized as a fusion polypeptide comprising: (a) a signalpeptide that promotes extracellular secretion of the soluble CD39 fromthe cell, the signal peptide being cleaved upon secretion, (b) one ormore amino acids added to the N-terminus of the soluble CD39 polypeptidein order to improve expression levels and/or stability, and (c) afragment of CD39 that possesses biological activity.

[0064] CD39 fusion polypeptides can also comprise polypeptides added toprovide novel polyfunctional entities. Further, soluble CD39-containingfusion polypeptides can comprise peptides added to facilitatepurification and identification of soluble CD39. Such peptides include,for example, poly-His or the antigenic identification peptides describedin U.S. Pat. No. 5,011,912 and in Hopp et al., Bio/Technology 6:1204,1988. One such peptide is the FLAG^(®) peptide,Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (SEQ ID NO:10), which is highlyantigenic and provides an epitope reversibly bound by a specificmonoclonal antibody, enabling rapid assay and facile purification ofexpressed recombinant polypeptide. A murine hybridoma designated 4E11produces a monoclonal antibody that binds the FLAG^(®) peptide in thepresence of certain divalent metal cations, as described in U.S. Pat.No. 5,011,912, hereby incorporated by reference. The 4E11 hybridoma cellline has been deposited with the American Type Culture Collection underaccession no. HB 9259. Monoclonal antibodies that bind the FLAG^(®)peptide are available from Eastman Kodak Co., Scientific Imaging SystemsDivision, New Haven, Conn.

[0065] Another particularly useful class of fusion polypeptides includesthose that allow localization or concentration of CD39 at a site ofplatelet activation and recruitment. Such fusion polypeptides comprise amoiety that specifically binds activated platelets and CD39, and can beprepared using recombinant DNA technology, or by using standardtechniques for conjugation of polypeptides. For example, WO 95/12412discloses platelet-specific chimeric antibodies and methods of using thesame in treating various thrombotic disorders. These antibodies, orother platelet specific antibodies (for example, antibodies toP-selectin/CD62), are useful in forming fusion polypeptides with CD39.Moreover, humanized or single chain antibodies can be prepared, based onsuch platelet specific antibodies.

[0066] Counterstructure molecules (molecules that specifically bindpolypeptides expressed on the cell surface of activated platelets) andfragments thereof that bind to platelets are also useful in formingfusion polypeptides that bind specifically to activated platelets.Exemplary counterstructures include ligands for P-selectin/CD62 (see,i.e., Varki A., Proc Natl Acad Sci U S A 91:7390, 1994; Sammar et al.,Int Immunol 6:1027, 1994; Lenter et al., J Cell Biol 125:471, 1994).

[0067] Encompassed by the present invention are oligomers that containCD39 polypeptides. CD39 oligomers may be in the form ofcovalently-linked or non-covalently-linked multimers, including dimers,trimers, or higher oligomers. Oligomers may be linked by disulfide bondsformed between cysteine residues on different CD39 polypeptides.Alternatively, oligomers may be formed by constructing fusionpolypeptides of CD39 and the Fc region of an immunoglobulin molecule,such as human IgG₁, to yield a CD39/Fc fusion polypeptide. The term “Fcpolypeptide” as used herein includes native and mutein forms ofpolypeptides derived from the Fc region of an antibody. Truncated formsof such polypeptides containing the hinge region that promotesdimerization are also included. One suitable Fc polypeptide, describedin PCT application WO 93/10151 (hereby incorporated by reference), is asingle chain polypeptide extending from the N-terminal hinge region tothe native C-terminus of the Fc region of a human IgG1 antibody. Anotheruseful Fc polypeptide is the Fc mutein described in U.S. Pat. No.5,457,035 and in Baum et al., (EMBO J. 13:3992-4001, 1994). The aminoacid sequence of this mutein is identical to that of the native Fcsequence presented in WO 93/10151, except that amino acid 19 has beenchanged from Leu to Ala, amino acid 20 has been changed from Leu to Glu,and amino acid 22 has been changed from Gly to Ala. The mutein exhibitsreduced affinity for Fc receptors. The CD39/Fc fusion polypeptides areallowed to assemble much like heavy chains of an antibody molecule toform divalent CD39. If fusion polypeptides are made with both heavy andlight chains of an antibody, it is possible to form a CD39 oligomer withas many as four CD39 extracellular regions.

[0068] In some embodiments of the invention, oligomers comprisingmultiple CD39 polypeptides are joined via covalent or non-covalentinteractions between peptide moieties fused to the C39-polypeptides.Such peptide moieties may be peptide linkers (spacers), or peptides thathave the property of promoting oligomerization. Leucine zippers andcertain polypeptides derived from antibodies are among the peptides thatcan promote oligomerization of polypeptides.

[0069] The present invention comprises fusion polypeptides with orwithout spacer amino acid linking groups. For example, two soluble CD39domains can be linked with a linker sequence, such as(Gly)₄Ser(Gly)₅Ser, which is described in U.S. Pat. No. 5,073,627. Otherlinker sequences include, for example, GlyAlaGlyGlyAlaGlySer(Gly)₅Ser,(Gly₄Ser)₂, (GlyThrPro)₃, and (Gly₄Ser)₃Gly₄SerGly₅Ser. Alternatively,CD39 can be linked to another polypeptide (non-CD39) with or without aspacer amino acid linking group. As shown in Example 9, ThrSerSer orThrSerSerGly linkers may be used to fuse IL2 residues to soluble CD39.For the expression of soluble CD39, the inventors have made thesurprising and unexpected discovery that the fusion of 12 amino acidsfrom the N-terminus of mature human IL2 to the solCD39 coding region,results in high levels of both expression and activity in thesupernatants of transfected cells. Among the particularly preferredembodiments of the invention, therefore, are soluble CD39 polypeptideshaving an amino acid sequence SEQ ID NO:6 and nucleic acids, such as SEQID NO:5, that encode soluble CD39 polypeptides having an amino acidsequence SEQ ID NO:6.

[0070] The present invention further includes soluble CD39 polypeptideswith or without associated native-pattern glycosylation. CD39 expressedin yeast or mammalian expression systems (e.g., COS-7 cells) may besimilar to or significantly different from a native CD39 polypeptide inmolecular weight and glycosylation pattern, depending upon the choice ofexpression system. Expression of CD39 polypeptides in bacterialexpression systems, such as E. coli, provides non-glycosylatedmolecules.

[0071] Different host cells may process polypeptides differentially,resulting in heterogeneous mixtures of polypeptides with variable N- orC-termini. Expression of soluble CD39 polypeptides in microbialexpression systems, such as E. coli, generally provides a homogeneouspolypeptide preparation. Polypeptides may be differentially processed bya eukaryotic cell, resulting in variable N- and C-termini, and henceyield a heterogeneous polypeptide preparation. The present inventionincludes polypeptides, produced by eukaryotic host cells, which havevariable N-termini or C-termini. In one embodiment of the inventive CD39polypeptides, the amino and carboxy termini can be about five aminoacids different from those disclosed herein.

[0072] The skilled artisan will also recognize that the position(s) atwhich a signal peptide is cleaved may differ from that predicted bycomputer program, and may vary according to such factors as the type ofhost cells employed in expressing a recombinant soluble CD39polypeptide. A polypeptide preparation according to the invention maytherefore include a mixture of polypeptide molecules having differentN-terminal amino acids, resulting from cleavage of the signal peptide atmore than one site.

[0073] D. Nucleic Acids

[0074] The invention encompasses full length nucleic acid moleculesencoding soluble CD39 as well as isolated fragments and oligonucleotidesderived from the nucleotide sequence of SEQ ID NO:1. Such nucleic acidsequences may include nucleotides 178-1494 of SEQ ID NO:1 or a fragmentthereof, and DNA and/or RNA sequences that hybridize to the codingregion of the nucleotide sequence of SEQ ID NO:1, or its complement,under conditions of moderate stringency, and which encode polypeptidesor fragments thereof of the invention.

[0075] Nucleic acid sequences encoding soluble CD39 polypeptides havingaltered glycosylation sites, deleted or substituted Cys residues, ormodified proteolytic cleavage sites, nucleic acid sequences encodingsub-units of CD39 polypeptides or fusion polypeptides of CD39 with otherpeptides, allelic variants of CD39, mammalian homologs of CD39, andnucleic acid sequences encoding CD39 polypeptides derived fromalternative mRNA constructs, or those that encode peptide havingsubstituted or additional amino acids, are examples of nucleic acidsequences according to the invention.

[0076] Due to degeneracy of the genetic code, there can be considerablevariation in nucleotide sequences encoding the same amino acid sequence.Included as embodiments of the invention are sequences capable ofhybridizing under moderately stringent conditions (e.g., prewashingsolution of 5 X SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0) and hybridizationconditions of 50° C., 5 X SSC, overnight) to the DNA sequences encodingsoluble CD39, and other sequences which are degenerate to those whichencode soluble CD39. The skilled artisan can determine additionalcombinations of salt and temperature that constitute moderatehybridization stringency. Conditions of higher stringency include highertemperatures for hybridization and post-hybridization washes, and/orlower salt concentration.

[0077] In a preferred embodiment, CD39 DNAs include those that encodepolypeptides that are at least about 70% or at least 80% identical inamino acid sequence to the amino acid sequence of native CD39polypeptide as set forth in SEQ ID NO:1. In a more preferred embodiment,the encoded variants of CD39 are at least about 90% or at least about95% identical in amino acid sequence to the native form of CD39; in amost preferred embodiment, the encoded variants of CD39 are at leastabout 98% or at least about 99% identical in amino acid sequence to thenative form of CD39. For DNAs that encode a fragment of CD39, percentidentity of the fragment is based on percent identity to thecorresponding portion of full-length CD39.

[0078] Mutations can be introduced into nucleic acids by synthesizingoligonucleotides containing a mutant sequence, flanked by restrictionsites enabling ligation to fragments of the native sequence. Followingligation, the resulting reconstructed sequence encodes a variant havingthe desired amino acid insertion, substitution, or deletion.

[0079] Alternatively, oligonucleotide-directed site-specific mutagenesisprocedures can be employed to provide an altered gene having particularcodons altered according to the substitution, deletion, or insertionrequired. Exemplary methods of making the alterations set forth aboveare disclosed by Walder et al. (Gene 42:133, 1986); Bauer et al. (Gene37:73, 1985); Craik (BioTechniques, January 1985, 12-19); Smith et al.(Genetic Engineering: Principles and Methods, Plenum Press, 1981); andU.S. Pat. Nos. 4,518,584 and 4,737,462 disclose suitable techniques, andare incorporated by reference herein.

[0080] The well known polymerase chain reaction (PCR) procedure also maybe employed to generate and amplify a DNA sequence encoding a desiredpolypeptide or fragment thereof. Oligonucleotides that define thedesired termini of the DNA fragment are employed as 5′ and 3′ primers.The oligonucleotides may additionally contain recognition sites forrestriction endonucleases to facilitate insertion of the amplified DNAfragment into an expression vector. PCR techniques are described inSaiki et al., Science 239:487, 1988; Recombinant DNA Methodology, Wu etal., eds., Academic Press, Inc., San Diego, 1989, pp. 189-196; and PCRProtocols: A Guide to Methods and Applications, Innis et al., eds.,Academic Press, Inc., 1990.

[0081] DNA sequences that encode CD39 polypeptides comprising variousadditions or substitutions of amino acid residues or sequences, ordeletions of terminal or internal residues or sequences not needed forbiological activity can be prepared. For example, N-glycosylation sitescan be modified to preclude glycosylation while allowing expression of ahomogeneous, reduced carbohydrate variant using yeast expressionsystems. N-glycosylation sites in eukaryotic polypeptides arecharacterized by an amino acid triplet Asn-X-Y, wherein X is any aminoacid except Pro and Y is Ser or Thr. Appropriate modifications to thenucleotide sequence encoding this triplet will result in substitutions,additions or deletions that prevent attachment of carbohydrate residuesat the Asn side chain.

[0082] In another example, sequences encoding Cys residues can bealtered to cause the Cys residues to be deleted or replaced with otheramino acids, preventing formation of incorrect intramolecular disulfidebridges upon renaturation. Thus, Cys residues may be replaced withanother amino acid or deleted without affecting polypeptide tertiarystructure or disulfide bond formation.

[0083] Other approaches to mutagenesis involve modification of sequencesencoding dibasic amino acid residues to enhance expression in yeastsystems in which KEX2 protease activity is present. Other variants areprepared by modification of adjacent dibasic amino acid residues, toenhance expression in yeast systems in which KEX2 protease activity ispresent. EP 212,914 discloses the use of site-specific mutagenesis toinactivate KEX2 protease processing sites in a polypeptide. KEX2protease processing sites are inactivated by deleting, adding orsubstituting residues to alter Arg-Arg, Arg-Lys, and Lys-Arg pairs toeliminate the occurrence of these adjacent basic residues. Similarmodification may be made to sequences encoding sites recognized andcleaved by other proteolytic enzymes. Sub-units of a CD39 polypeptidemay be constructed by deleting sequences encoding terminal or internalresidues or sequences not necessary for biological activity. Sequencesencoding fusion polypeptides as described below may be constructed byligating sequences encoding additional amino acid residues to theinventive sequences without affecting biological activity.

[0084] Mutations in nucleotide sequences constructed for expression of asoluble CD39 must, of course, preserve the reading frame phase of thecoding sequences and preferably will not create complementary regionsthat could hybridize to produce secondary mRNA structures such as loopsor hairpins which would adversely affect translation of the receptormRNA. Although a mutation site may be predetermined, it is not necessarythat the nature of the mutation per se be predetermined. For example, inorder to select for optimum characteristics of mutants at a given site,random mutagenesis may be conducted at the target codon and theexpressed mutated polypeptides screened for the desired activity.

[0085] Not all mutations in the nucleotide sequence which encodes a CD39polypeptide will be expressed in the final product, for example,nucleotide substitutions may be made to enhance expression, primarily toavoid secondary structure loops in the transcribed mRNA (see EPA75,444A, incorporated herein by reference), or to provide codons thatare more readily translated by the selected host, e.g., the well-knownE. coli preference codons for E. coli expression.

[0086] In the genome, CD39 polypeptides are encoded by multi-exon genes.The present invention further includes alternative MRNA constructs whichcan be attributed to different mRNA splicing events followingtranscription and which hybridize with the cDNAs disclosed herein underconditions of moderate stringency. CD39 polypeptides according to theinvention include allelic variations of the sequence shown in SEQ IDNO:1, and sequences encoding CD39 polypeptides that comprise additionalamino acids to those of SEQ ID NO:1.

[0087] The isolated nucleic acid sequences of this invention aresufficiently free of association with nucleic acid sequences encodingother proteinaceous material, and from other materials found in livingcells, such as proteins, lipids or carbohydrates, to allow the skilledartisan to prepare vectors for the expression of soluble CD39polypeptides.

[0088] E. Recombinant Expression Systems

[0089] The present invention also provides recombinant cloning andexpression vectors containing CD39 DNA, as well as host cells containingthe recombinant vectors. Expression vectors comprising CD39 DNA may beused to prepare soluble CD39 polypeptides encoded by the DNA. Theexpression vectors carrying the recombinant CD39 DNA sequence aretransferred, for example by transfection or transformation, into asubstantially homogeneous culture of a suitable host microorganism ormammalian cell line. Transformed host cells are cells which have beentransformed or transfected with nucleotide sequences encoding CD39polypeptides and express CD39 polypeptides. Expressed CD39 polypeptideswill be located within the host cell and/or secreted into culturesupernatant fluid, depending upon the nature of the host cell and thegene construct inserted into the host cell. The skilled artisan willrecognize that the procedure for purifying the expressed CD39 will varyaccording to such factors as the type of host cells employed.

[0090] Any suitable expression system may be employed. Recombinantexpression vectors for expression of soluble CD39 by recombinant DNAtechniques include a CD39 DNA sequence comprising a synthetic orcDNA-derived DNA fragment encoding a CD39 polypeptide, operably linkedto a suitable transcriptional or translational regulatory nucleotidesequence, such as one derived from a mammalian, microbial, viral, orinsect gene.

[0091] Examples of regulatory sequences include transcriptionalpromoters, operators, or enhancers, an mRNA ribosomal binding site, andappropriate sequences which control transcription and translationinitiation and termination. Nucleotide sequences are operably linkedwhen the regulatory sequence functionally relates to the CD39 DNAsequence. Thus, a promoter nucleotide sequence is operably linked to aCD39 DNA sequence if the promoter nucleotide sequence controls thetranscription of the CD39 DNA sequence. An origin of replication thatconfers the ability to replicate in the desired host cells, and aselection gene by which transformants are identified, are generallyincorporated into the expression vector.

[0092] In addition, a sequence encoding an appropriate signal peptide(native or heterologous) can be incorporated into expression vectors. ADNA sequence for a signal peptide (secretory leader) may be fused inframe to the CD39 sequence so that the CD39 is initially translated as afusion polypeptide comprising the signal peptide. A signal peptide thatis functional in the intended host cells promotes extracellularsecretion of the CD39 polypeptide. The signal peptide is cleaved fromthe CD39 polypeptide upon secretion of soluble CD39 from the cell.

[0093] Regarding signal peptides that may be employed in producingsoluble CD39, the native signal peptide may be replaced by aheterologous signal peptide or leader sequence, if desired. The choiceof signal peptide or leader may depend on factors such as the type ofhost cells in which the recombinant polypeptide is to be produced. Toillustrate, examples of heterologous signal peptides that are functionalin mammalian host cells include the signal sequence for interleukin-7(IL-7) described in U.S. Pat. No. 4,965,195, the signal sequence forinterleukin-2 receptor described in Cosman et al., Nature 312:768, 1984;the interleukin-4 receptor signal peptide described in EP 367,566; thetype I interleukin-1 receptor signal peptide described in U.S. Pat. No.4,968,607; and the type II interleukin-1 receptor signal peptidedescribed in EP 460,846. For the expression of soluble CD39, theinventors have made the surprising and unexpected discovery that the useof a leader containing sequences derived from a human IL-2 polypeptide(SEQ ID NO:9) results in high levels of ATPase activity in thesupernatants of transfected cells. Among the particularly preferredembodiments of the invention, therefore, are nucleic acids encodingsoluble CD39 polypeptides having an amino acid sequence SEQ ID NO:8.

[0094] Suitable host cells for expression of CD39 polypeptides includeprokaryotes, yeast or higher eukaryotic cells. Mammalian or insect cellsare generally preferred for use as host cells. Appropriate cloning andexpression vectors for use with bacterial, fungal, yeast, and mammaliancellular hosts are described, for example, in Pouwels et al. CloningVectors: A Laboratory Manual, Elsevier, N.Y., 1985. Cell-freetranslation systems could also be employed to produce soluble CD39polypeptides using RNAs derived from DNA constructs disclosed herein.

[0095] Prokaryotes include gram negative or gram positive organisms, forexample, E. coli or Bacilli. Suitable prokaryotic host cells fortransformation include, for example, E. coli, Bacillus subtilis,Salmonella typhimurium, and various other species within the generaPseudomonas, Streptomyces, and Staphylococcus. In a prokaryotic hostcell, such as E. coli, polypeptides may include an N-terminal methionineresidue to facilitate expression of the recombinant polypeptide in theprokaryotic host cell. The N-terminal Met may be cleaved from theexpressed recombinant polypeptide.

[0096] Expression vectors for use in prokaryotic host cells generallycomprise one or more phenotypic selectable marker genes. A phenotypicselectable marker gene is, for example, a gene encoding a protein thatconfers antibiotic resistance or that supplies an autotrophicrequirement. Examples of useful expression vectors for prokaryotic hostcells include those derived from commercially available plasmids such asthe cloning vector pBR322 (ATCC 37017). pBR322 contains genes forampicillin and tetracycline resistance and thus provides simple meansfor identifying transformed cells. An appropriate promoter and a CD39DNA sequence are inserted into the pBR322 vector. Other commerciallyavailable vectors include, for example, pKK223-3 (Pharmacia FineChemicals, Uppsala, Sweden) and pGEM1 (Promega Biotec, Madison, Wiss.,USA).

[0097] Promoter sequences commonly used for recombinant prokaryotic hostcell expression vectors include β-lactamase (penicillinase), lactosepromoter system (Chang et al., Nature 275:615, 1978; and Goeddel et al.,Nature 281:544, 1979), tryptophan (trp) promoter system (Goeddel et al.,Nucl. Acids Res. 8:4057, 1980; and EP-A-36776) and tac promoter(Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory, p. 412, 1982). A particularly useful prokaryotic host cellexpression system employs a phage λ P_(L) promoter and a cI857tsthermolabile repressor sequence. Plasmid vectors available from theAmerican Type Culture Collection which incorporate derivatives of the λP_(L) promoter include plasmid pHUB2 (resident in E. coli strain JMB9,ATCC 37092) and pPLc28 (resident in E. coli RR1, ATCC 53082).

[0098] Soluble CD39 may also be expressed in yeast host cells,preferably from the Saccharomyces genus (e.g., S. cerevisiae). Othergenera of yeast, such as Pichia or Kluyveromyces, may also be employed.Yeast vectors will often contain an origin of replication sequence froma 2μ yeast plasmid, an autonomously replicating sequence (ARS), apromoter region, sequences for polyadenylation, sequences fortranscription termination, and a selectable marker gene. Suitablepromoter sequences for yeast vectors include, among others, promotersfor metallothionein, 3-phosphoglycerate kinase (Hitzeman et al., J.Biol. Chem. 255:2073, 1980) or other glycolytic enzymes (Hess et al., J.Adv. Enzyme Reg. 7:149, 1968; and Holland et al., Biochem. 17:4900,1978), such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phospho-glucose isomerase, andglucokinase. Other suitable vectors and promoters for use in yeastexpression are further described in Hitzeman, EPA-73,657. Anotheralternative is the glucose-repressible ADH2 promoter described byRussell et al. (J. Biol. Chem. 258:2674, 1982) and Beier et al. (Nature300:724, 1982). Shuttle vectors replicable in both yeast and E. coli maybe constructed by inserting DNA sequences from pBR322 for selection andreplication in E. coli (Amp^(r) gene and origin of replication) into theabove-described yeast vectors.

[0099] The yeast α-factor leader sequence may be employed to directsecretion of recombinant polypeptides. The α-factor leader sequence isoften inserted between the promoter sequence and the structural genesequence. See, e.g., Kurjan et al., Cell 30:933, 1982 and Bitter et al.,Proc. Natl. Acad. Sci. USA 81:5330, 1984. Other leader sequencessuitable for facilitating secretion of recombinant polypeptides fromyeast hosts are known to those of skill in the art. A leader sequencemay be modified near its 3′ end to contain one or more restrictionsites. This will facilitate fusion of the leader sequence to thestructural gene.

[0100] Yeast transformation protocols are known to those of skill in theart. One such protocol is described by Hinnen et al., Proc. Natl. Acad.Sci. USA 75:1929, 1978. The Hinnen et al. protocol selects for Trp⁺transformants in a selective medium, wherein the selective mediumconsists of 0.67% yeast nitrogen base, 0.5% casamino acids, 2% glucose,10 μg/ml adenine and 20 μg/ml uracil.

[0101] Yeast host cells transformed by vectors containing an ADH2promoter sequence may be grown for inducing expression in a “rich”medium. An example of a rich medium is one consisting of 1% yeastextract, 2% peptone, and 1% glucose supplemented with 80 μg/ml adenineand 80 μg/ml uracil. Derepression of the ADH2 promoter occurs whenglucose is exhausted from the medium.

[0102] Mammalian or insect host cell culture systems also may beemployed to express recombinant CD39 polypeptides. Bacculovirus systemsfor production of heterologous polypeptides in insect cells are reviewedby Luckow and Summers, Bio/Technology 6:47, 1988. Established cell linesof mammalian origin may also be used. Examples of suitable mammalianhost cell lines include the COS-7 line of monkey kidney cells (ATCC CRL1651) (Gluzman et al., Cell 23:175, 1981), L cells, C127 cells, 3T3cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells, HeLa cells, andBHK (ATCC CRL 10) cell lines, and the CV1/EBNA cell line derived fromthe African green monkey kidney cell line CV1 (ATCC CCL 70) as describedby McMahan et al. (EMBO J. 10: 2821, 1991). For the production oftherapeutic polypeptides it is particularly advantageous to use amammalian host cell line which has been adapted to grow in media thatdoes not contain animal proteins. The use of such a cell line for theexpression of soluble CD39 is described in Example 13.

[0103] Established methods for introducing DNA into mammalian cells havebeen described (Kaufman, R. J., Large Scale Mammalian Cell Culture,1990, pp. 15-69). Additional protocols using commercially availablereagents, such as Lipofectamine (Gibco/BRL) or Lipofectamine-Plus, canbe used to transfect cells (Felgner et al., Proc. Natl. Acad. Sci. USA84:7413-7417, 1987). In addition, electroporation can be used totransfect mammalian cells using conventional procedures, such as thosein Sambrook et al. Molecular Cloning: A Laboratory Manual, 2 ed. Vol.1-3, Cold Spring Harbor Laboratory Press, 1989). Selection of stabletransformants can be performed using methods known in the art, such as,for example, resistance to cytotoxic drugs. Kaufman et al., Meth. inEnzymology 185:487-511, 1990, describes several selection schemes, suchas dihydrofolate reductase (DHFR) resistance. A suitable host strain forDHFR selection can be CHO strain DX-B11, which is deficient in DHFR(Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77:4216-4220, 1980). Aplasmid expressing the DHFR cDNA can be introduced into strain DX-B11,and only cells that contain the plasmid can grow in the appropriateselective media. Other examples of selectable markers that can beincorporated into an expression vector include cDNAs conferringresistance to antibiotics, such as G418 and hygromycin B. Cellsharboring the vector can be selected on the basis of resistance to thesecompounds.

[0104] Transcriptional and translational control sequences for mammalianhost cell expression vectors can be excised from viral genomes. Commonlyused promoter sequences and enhancer sequences are derived from polyomavirus, adenovirus 2, simian virus 40 (SV40), and human cytomegalovirus.DNA sequences derived from the SV40 viral genome, for example, SV40origin, early and late promoter, enhancer, splice, and polyadenylationsites can be used to provide other genetic elements for expression of astructural gene sequence in a mammalian host cell. Viral early and latepromoters are particularly useful because both are easily obtained froma viral genome as a fragment, which can also contain a viral origin ofreplication (Fiers et al., Nature 273:113, 1978; Kaufman, Meth. inEnzymology, 1990). Smaller or larger SV40 fragments can also be used,provided the approximately 250 bp sequence extending from the Hind IIIsite toward the Bgl I site located in the SV40 viral origin ofreplication site is included.

[0105] Additional control sequences shown to improve expression ofheterologous genes from mammalian expression vectors include suchelements as the expression augmenting sequence element (EASE) derivedfrom CHO cells (Morris et al., Animal Cell Technology, 1997, pp.529-534) and the tripartite leader (TPL) and VA gene RNAs fromAdenovirus 2 (Gingeras et al., J. Biol. Chem. 257:13475-13491, 1982).The internal ribosome entry site (IRES) sequences of viral origin allowsdicistronic mRNAs to be translated efficiently (Oh and Sarnow, CurrentOpinion in Genetics and Development 3:295-300, 1993; Ramesh et al.,Nucleic Acids Research 24:2697-2700, 1996). Expression of a heterologouscDNA as part of a dicistronic mRNA followed by the gene for a selectablemarker (e.g. DHFR) has been shown to improve transfectability of thehost and expression of the heterologous cDNA (Kaufman, Meth. inEnzymology, 1990). Exemplary expression vectors that employ dicistronicmRNAs are pTR-DC/GFP described by Mosser et al., Biotechniques22:150-161, 1997, and p2A5I described by Morris et al., Animal CellTechnology, 1997, pp. 529-534.

[0106] A useful high expression vector, pCAVNOT, has been described byMosley et al., Cell 59:335-348, 1989. Other expression vectors for usein mammalian host cells can be constructed as disclosed by Okayama andBerg (Mol. Cell. Biol. 3:280, 1983). A useful system for stable highlevel expression of mammalian cDNAs in C127 murine mammary epithelialcells can be constructed substantially as described by Cosman et al.(Mol. Immunol. 23:935, 1986). A useful high expression vector, PMLSVN1/N4, described by Cosman et al., Nature 312:768, 1984, has beendeposited as ATCC 39890. Additional useful mammalian expression vectorsare described in EP-A-0367566, and in U.S. patent application Ser. No.07/701,415, filed May 16, 1991, incorporated by reference herein. Thevectors can be derived from retroviruses. In place of the native signalsequence, a heterologous signal sequence can be added, such as thesignal sequence for IL-7 described in U.S. Pat. No. 4,965,195; thesignal sequence for IL-2 receptor described in Cosman et al., Nature312:768, 1984; the IL-4 signal peptide described in EP 367,566; the typeI IL-1 receptor signal peptide described in U.S. Pat. No. 4,968,607; andthe type II IL-1 receptor signal peptide described in EP 460,846.

[0107] Another useful expression vector, pFLAG, can be used. FLAG^(®)technology is centered on the fusion of a low molecular weight (1kD),hydrophilic, FLAG^(®) marker peptide to the N-Terminus of a recombinantpolypeptide expressed by the pFLAG-1^(™) Expression Vector (obtainedfrom IBI Kodak).

[0108] F. Purification of soluble CD39 Polypeptides

[0109] Soluble CD39 polypeptides may be prepared by culturingtransformed host cells under culture conditions necessary to expressCD39 polypeptides. The resulting expressed polypeptides may then bepurified from culture media or cell extracts. Supernatant fluid from thecultured, transformed host cells may be concentrated using acommercially available protein concentration filter, for example, anAmicon or Millipore Pellicon ultrafiltration unit. Following theconcentration step, the concentrate may be applied to a cation exchangematrix. Suitable cation exchangers include various insoluble matricescomprising sulfonic or carboxymethyl groups; sulfonic groups arepreferred. The matrices can be acrylamide, agarose, dextran, celluloseor other types commonly employed in protein purification. Subsequently,an anion exchange resin is employed, for example, a matrix or substratehaving pendant diethylaminoethyl (DEAE) or quaternary amino groups;quaternary amino groups are preferred. The matrices can be acrylamide,agarose, dextran, cellulose or other types commonly employed in proteinpurification. Additionally, a gel filtration medium may be employed tofurther purify CD39 polypeptides according to approximate molecularweight. Alternatively, certain of these steps may not be performed, ormay be performed in the reverse order.

[0110] One or more reverse-phase high performance liquid chromatography(RP-HPLC) steps employing hydrophobic RP-HPLC media, (e.g., silica gelhaving pendant methyl or other aliphatic groups) may be employed tofurther purify CD39. A substantially purified and homogeneouspolypeptide having CD39 biological activity may be eluted from apolyacrylamide gel subsequent to electrophoretic separation. Some or allof the foregoing purification steps, in various combinations, can alsobe employed to provide a substantially purified and homogeneousrecombinant polypeptide containing less than about 1% by mass of proteincontaminants residual of production processes, or alternatively, whichis greater than about 95% pure by gel electrophoresis.

[0111] Affinity chromatography may be utilized to purify soluble CD39.Affinity purification of soluble CD39 from conditioned media isdescribed in Example 12C. Moreover, small amounts of purified CD39 maybe obtained by immunoprecipitating CD39 with a monoclonal antibody,electrophoresing the immunoprecipitate on a polyacrylamide gel, excisingthe portion of the gel containing the CD39, and eluting the CD39 fromthe excised portion of the gel.

[0112] Recombinant polypeptides produced in bacterial culture aregenerally isolated by disruption of the host cells, centrifugation,extraction from cell pellets if an insoluble polypeptide, or from thesupernatant fluid if a soluble polypeptide, followed by one or moreconcentration, salting-out, ion exchange, affinity purification or sizeexclusion chromatography steps. Finally, RP-HPLC can be employed forfinal purification steps. Microbial cells can be disrupted by anyconvenient method, including freeze-thaw cycling, sonication, mechanicaldisruption, or use of cell lysing agents.

[0113] Transformed yeast host cells may be employed to express CD39 as asecreted polypeptide. This simplifies purification. Secreted recombinantpolypeptide from a yeast host cell fermentation can be purified bymethods analogous to those disclosed by Urdal et al. (J. Chromatog.296:171, 1984). Urdal et al. describe two sequential, reversed-phaseHPLC steps for purification of recombinant human IL-2 on a preparativeHPLC column.

[0114] The desired degree of purity of soluble CD39 polypeptides dependson the intended use of the polypeptide. A relatively high degree ofpurity is desired when the polypeptide is to be administered in vivo,for example. Advantageously, soluble CD39 polypeptides are purified suchthat no protein bands corresponding to other (non-CD39) polypeptides aredetectable upon analysis by SDS-polyacrylamide gel electrophoresis(SDS-PAGE). Following electrophoresis, the protein band may bevisualized by silver staining, Coomassie blue staining, or (if theprotein is radiolabeled) by autoradiography. It will be recognized byone skilled in the pertinent field that multiple bands corresponding toCD39 polypeptide may be visualized by SDS-PAGE, due to differentialglycosylation, differential post-translational processing, and the like.

[0115] G. Therapeutic Compositions of CD39 Polypeptides

[0116] The present invention provides compositions comprising aneffective amount of a soluble CD39 polypeptide in a pharmaceuticallyacceptable carrier. As used herein, the terms “therapy,” “therapeutic,”“treat,” and “treatment” generally include prophylaxis, i.e.,prevention, of a disease or condition in addition to therapy ortreatment for an extant disease or condition. Therapeutic compositionsof soluble CD39 polypeptides may therefore need to be administeredbefore, during, or after the presentation of symptoms. For therapeuticuse, a soluble CD39 polypeptide is administered to a patient fortreatment in a manner appropriate to the indication. Thus, for example,soluble CD39 pharmaceutical compositions which are administered toachieve a desired therapeutic effect can be given by bolus injection,continuous infusion, sustained release from implants or the like, orother suitable technique. Ideally, development of a stable form of CD39or closely related biologically active variant would allow its use inoral form, a preferable route of administration. Since CD39 isaspirin-insensitive, these two therapeutic agents (CD39 compositions andaspirin) can be used in combination, for maximal benefit.

[0117] Typically, a soluble CD39 therapeutic agent will be administeredin the form of a pharmaceutical composition comprising purified solubleCD39 in conjunction with physiologically acceptable carriers, includingexcipients or diluents. Such carriers will be nontoxic to patients atthe dosages and concentrations employed. As described in the examplesthat follow, the administration of CD39 in murine and porcine models ofthrombosis does not cause any observable toxic effects. Moreover, asecond dose of CD39 does not evoke any signs of immunogenicity.Ordinarily, the preparation of such compositions entails combining asoluble CD39 composition with buffers, antioxidants such as ascorbicacid, low molecular weight (less than about 10 residues) polypeptides,polypeptides, amino acids, carbohydrates including glucose, sucrose ordextrans, chelating agents such as EDTA, glutathione and otherstabilizers and excipients. Neutral buffered saline or saline mixed withconspecific serum albumin are exemplary appropriate diluents.

[0118] One type of sustained release technology which may be used inadministering soluble CD39 compositions is that utilizing hydrogelmaterials, for example, photopolymerizable hydrogels (Sawhney et al.,Macromolecules 26:581; 1993). Similar hydrogels have been used toprevent postsurgical adhesion formation (Hill-West et al., Obstet.Gynecol. 83:59, 1994) and to prevent thrombosis and vessel narrowingfollowing vascular injury (Hill-West et al., Proc. Natl. Acad. Sci. USA91:5967, 1994). Polypeptides can be incorporated into such hydrogels toprovide sustained, localized release of active agents (West and Hubbel,Reactive Polymers 25:139, 1995; Hill-West et al., J. Surg. Res. 58:759;1995). The sustained, localized release of CD39 when incorporated intohydrogels would be amplified by the long half life of CD39, which isdemonstrated in the Examples below.

[0119] Accordingly, the soluble CD39 compositions described herein canalso be incorporated into hydrogels, for application to tissues forwhich localized inhibition of hemostasis is desirable. For example, ahydrogel incorporating a CD39 polypeptide can be applied to tissue aftersurgery, to prevent or reduce post-surgical adhesion formation, or canbe applied using a catheter-based delivery system following angioplastyto prevent or reduce restenosis. Those of skill in the art will be ableto formulate an appropriate hydrogel by applying standardpharmacokinetic studies, for example as discussed in West and Hubbell,supra.

[0120] Effective amounts may vary, depending on the age, type andseverity of the condition to be treated, body weight, desired durationof treatment, method of administration, and other parameters. Effectivedosages are determined by a physician or other qualified medicalprofessional. Typical dosages are 0.01-100 mg/kg body weight, preferably0.1-10 mg/kg body weight. In some embodiments a single administration issufficient; in some embodiments the soluble CD39 polypeptide isadministered on a daily basis for up to a week or as much as a month ormore.

[0121] The biological effectiveness of soluble CD39 polypeptides iseasily evaluable: at given time intervals after administration, aprolongation of the bleeding time in the setting of unchanged plateletcount should be measurable if released platelet ADP has been metabolizedby the CD39 composition administered. This would indicate that atherapeutic effect has likely been obtained, as said measurementcorrelates with clinical improvement. A therapeutic effect can also bevalidated by testing platelet reactivity to ADP and other plateletagonists ex vivo. Actual measurements of enzyme (apyrase) activity canalso be made following administration of soluble CD39. These and othermethods of measuring biological effectiveness are illustrated in theExamples below.

[0122] H. Abbreviations Used in the Specification

[0123] ACR, apyrase conserved regions;

[0124] AG, Affigel beads;

[0125] ASA, acetylsalicylic acid;

[0126] ATPDase, ATP diphosphohydolase;

[0127] CHO, Chinese hamster ovary;

[0128] CM, conditioned medium;

[0129] DHFR, dihydrofolate reductase;

[0130] FSBA, fluorosulfonylbenzoyl-adenosine;

[0131] HUVEC, human umbilical vein endothelial cells;

[0132] PRP, platelet-rich plasma;

[0133] PTCA, percutaneous transluminal coronary angioplasty;

[0134] solCD39, recombinant soluble human CD39;

[0135] TBS, Tris-buffered saline

EXAMPLES

[0136] The following examples are intended to illustrate particularembodiments and not to limit the scope of the invention.

Example 1 Assay For CD39 Expression

[0137] This example describes the use of a monoclonal antibody in a FACSassay to analyze expression of CD39. The B73 mAb, a monoclonalanti-CD39, is a murine IgG1 that was derived from BALB/c mice immunizedwith the RPMI 1788 cell line (Rector et al., Immunology 55:481, 1985)and characterized as CD39-specific by flow cytometric analysis andimmunoprecipitation/SDS-PAGE. Monoclonal anti-CD39 is purified fromascites fluid by affinity chromatography using a protein A column,eluted with 0.05 M sodium citrate, pH 3.0, neutralized and stored a 4°C. at a concentration of about 1 mg/ml.

[0138] Cells to be analyzed (e.g., MP-1 cells, U937 cells, U937 cellsstimulated with 5 ng/ml phorbol myristate acetate (PMA), or Daudi cells)are suspended to a concentration of 10⁶ cell in 50 μl of phosphatebuffered saline (PBS) containing 100 μg/ml human IgG1, and incubated for30 minutes. The cells are then pelleted by centrifugation, resuspendedin PBS/azide containing a first antibody (anti-CD39 or control antibody)and incubated (i.e., for 30 minutes at 4° C.) The cells are then washedtwo times in PBS/azide, resuspended, and incubated with a labeled secondantibody, for example, goat anti-murine immunoglobulin conjugated tophycoerythrin, then washed again. The cells are analyzed by flowcytometry, and levels of CD39 determined.

Example 2 Immunoselection of Cells Expressing CD39

[0139] This example describes a panning (immunoselection) technique forcells expressing CD39. For the preparation of pan plates, purifiedanti-CD39 or control antibody is diluted in phosphate buffered salinecontaining 0.1% heat-inactivated fetal calf serum (PBS/FCS). A titrationof anti-CD39 can be performed to determine the most effectiveconcentration of anti-CD39. Pan plates are prepared by adding three mlof antibody solution or PBS/FCS alone to each plate. The plates areincubated for approximately one hour at room temperature, washed fivetimes with PBS/FCS, and three ml of PBS/FCS containing 0.02% sodiumazide are added to each plate.

[0140] The cells to be analyzed (e.g., MP-1, U937, or Daudi cells) aresuspended in PBS/500 μM EDTA/0.02% sodium azide (PEA) containing 5% goatserum, 5% rabbit serum and 100 μg/ml human IgG₁, to a concentration of2×10⁶ cells/ml; 500 μl of each cell suspension is added to the preparedpan plates. The pan plates are incubated with the cell suspension forapproximately two hours at room temperature, then the plates are washedgently three times with PEA containing 10% FCS (PEA/FCS), and threetimes with PEA. The plates are examined with a microscope, and therelative number of cells bound to each plate is determined.

Example 3 cDNA Library Construction

[0141] This example describes preparation of a cDNA library from a humanB cell line referred to as MP-1, for expression cloning of human CD39.

[0142] The library construction techniques were substantially similar tothat described by Ausubel et al., eds., Current Protocols In MolecularBiology, Vol. 1, 1987. Briefly, total RNA was extracted from 8Mguanidine HC1-lysed MP-1 cell cultures using differential ethanolprecipitation and poly (A)⁺ mRNA was isolated and enriched by oligo dTcellulose chromatography. Double-stranded cDNA was made from an RNAtemplate substantially as described by Gubler et al., Gene 25:263, 1983.Poly(A)⁺ mRNA fragments were converted to RNA-cDNA hybrids using reversetranscriptase primed with random hexanucleotides. The RNA-cDNA hybridswere then converted into double-stranded cDNA fragments using RNAase Hin combination with DNA polymerase I. The resulting double-stranded cDNAwas blunt-ended with T4 DNA polymerase.

[0143] Unkinased (i.e. unphosphorylated) BglII adaptors were ligated to5′ ends of the above blunt-ended cDNA duplexes, using the adaptorcloning method described in Haymerle et al., Nucleic Acids Res. 14:8615,1986. Under the described conditions, only the 24-mer oligonucleotide(top strand) will covalently bond to the cDNA during the ligationreaction. The non-covalently bound adaptors (including the complementary20-mer oligonucleotide described above and any unligated adaptors) wereremoved by gel filtration chromatography at 65° C., leaving 24nucleotide non-self-complementary overhangs on the cDNA termini.

[0144] The adaptored cDNA was inserted into adaptored pDC303, amammalian expression vector that also replicates in E. coli. pDC303 wasassembled from pDC201 (a derivative of pMLSV, previously described byCosman et al., Nature 312: 768, 1984), SV40 and cytomegalovirus DNA andcomprises, in sequence with the direction of transcription from theorigin of replication, the following components: (1) SV40 sequences fromcoordinates 5171-270 containing the origin of replication, enhancersequences and early and late promoters; (2) cytomegalovirus promoter andenhancer regions (nucleotides 671-63 from the sequence published byBoechart et al. (Cell 41:521, 1985); (3) adenovirus-2 from coordinates5779-6079 containing the first exon of the tripartite leader (TPL),segment 7101-7172 and 9634-9693 containing the second exon and part ofthe third exon of the TPL and a multiple cloning site (MCS) containingsites for XhoI, KpnI, SmaI and BglI; (4) SV40 segments from coordinates4127-4100 and 2770-2533 containing the polyadenylation and terminationsignals for early transcription; (5) adenovirus-2 sequences fromcoordinates 10532-11156 of the virus-associated RNA genes VAI and VAIIof pDC201; and (6) pBR322 sequences from coordinates 4363-2486 and1094-375 containing the ampicillin resistance gene and origin ofreplication.

[0145] The MP-1 cDNA library in pDC303 was introduced into E. colistrain DH10B by electroporation. Recombinants were plated to provideapproximately 5,000 colonies per plate. These recombinants were pooledto give a bulk stock of approximately 500,000 recombinants forscreening. DNA was prepared from transformed bacteria and isolated bycesium chloride centrifugation.

Example 4 Molecular Cloning of Human CD39 cDNA

[0146] This example describes the isolation of a DNA molecule encodingCD39 from the expression cloning library described in Example 3.

[0147] A. Round I: Transfection and Immunoselection

[0148] The isolated plasmid DNA was transfected into a sub-confluentlayer of COS-7 cells using DEAE-dextran and a chloroquine treatmentsubstantially according to the procedures described in McMahan et al.,EMBO J. 10:2821; 1991.

[0149] COS-7 cells were maintained in transfection and growth medium(Dulbecco's modified Eagles′ medium containing 10% (v/v) fetal calfserum, 50 U/ml penicillin, 50 μg/ml streptomycin, 2 mM L-glutamine and50 μg/ml gentamicin) and were plated to a density of approximately1.5×10⁶ cells in 10 ml transfection and growth medium in 10 cm dishes.Medium was removed from adherent cells growing in a layer toapproximately 70% confluency, and replaced with 10 ml complete mediumcontaining 66.5 μM chloroquine. About 500 μl of a DNA solution (5 μgDNA, 0.5 mg/ml DEAE-dextran in transfection and growth medium containing66.5 μM chloroquine) was added to the cells and the mixture wasincubated at 37° C. in 10% CO₂ for about five hours.

[0150] Following incubation, media was removed and the cells wereshocked by addition of 5 ml transfection and growth medium containing10% DMSO (dimethylsulfoxide) for 2.5-20 minutes. Shocking was followedby replacement of the solution with 10 ml fresh transfection and growthmedium. Twelve plates of cells were grown in culture for two to threedays to permit transient expression of the inserted DNA sequences. Thecells were trypsinized after about 24 hours of growth in order to removethem from the plates. After an additional one to two days, cellsexpressing CD39 were selected by panning, essentially as described inExample 2. The cells were incubated in the mAb 73 pan plates for twohours at room temperature, after which unbound cells were removed bygently rinsing three times with PEA/FCS, then three times with PEA.

[0151] The cells that were not removed by rinsing were expressing CD39;cells expressing CD39 were lysed by the addition of 700 μl lysing buffercontaining sodium dodecyl sulfate (SDS) and incubation for 20 minutes atroom temperature. Lysates were transferred from each dish to individualmicrofuge tubes containing 100 μl of 5 M NaCl. The tubes were capped,mixed thoroughly by inverting about 20 times, and stored at 4° C.overnight. After overnight incubation at 4° C., high molecular weightDNA (debris) was removed by centrifugation, and 2 μg of glycogen wasadded to each supernatant. The supernatants were then extracted twicewith phenol/chloroform and once with chloroform/isoamyl alcohol. DNA wasethanol precipitated, washed with 80% ethanol, and vacuum dried. Thepurified DNA was then electroporated into E. coli, which were thenplated out on ampicillin plates. A large-scale transformation wascarried out in this manner, yielding a total of approximately 48,000colonies (sub-library 1). DNA was prepared from the colonies using CsCl;frozen stocks of the colonies were prepared at the same time.

[0152] B. Round II: Electroporation and Immunoselection

[0153] The DNA from sub-library 1 was electroporated into COS cells(10×10 cm plates). Transfected COS cells were incubated, harvested andpanned substantially as described for Round I above. DNA was isolatedand a sub-library (sub-library 2) of approximately 50,000 independentcolonies was prepared substantially as described above.

[0154] C. Round III: Electroporation and FACS Selection

[0155] The DNA from sub-library 2 was electroporated into COS cells(10×10 cm plates). Transfected COS cells were incubated and harvestedsubstantially as described for Round I above. The harvested cells wereanalyzed by FACS substantially as described in Example 1 above. A smallsubpopulation of cells expressing CD39 was observed, and was sorted outfrom the larger mixture of cells; DNA was isolated from the sortedcells. A sub-library (sub-library 3) of approximately 5,000 independentcolonies was prepared. The DNA was pooled into 10 pools of approximately500 colonies each; isolated DNA and frozen stocks of bacteria wereprepared for each pool.

[0156] D. Round IV: Transfection and Immunoselection

[0157] The DNA from sub-library 3 was transfected into COS cells usingDEAE-dextran and chloroquine treatment, and incubated, substantially asdescribed for Round I above, except that the cells were incubated onfibronectin-treated, chambered slides (10 slides, 1 for each pool, and 4control slides) instead of 10 cm plates. After two days of growth, thecells were harvested as described, and analyzed by FACS substantially asdescribed in Example 1 above, as well as by a slide dipping technique.In the slide dipping technique, the slides were incubated with¹²⁵I-labeled mAb 73 and fixed with glutaraldehyde. The results weredetermined by autoradiography using light microscopy to detect cellcontaining silver granules.

[0158] Two pools containing approximately 500 individual clones eachwere identified as potentially positive for production of CD39. Thepools were titered and plated to provide plates containing an average ofapproximately 150 colonies each. A replicate nitrocellulose filter wasprepared from each plate; each plate was then scraped to provide smallerpools of plasmid DNA.

[0159] E. Round V: Transfection and Immunoselection

[0160] COS-7 cells were transfected with the DNA from the smaller poolsby DEAE-dextran, according to the same procedure described above. Thetransfected cells were screened by slide dipping and FACS as describedpreviously. Two of the smaller pools contained clones that were positivefor CD39 as indicated by the presence of an expressed gene product thatbound mAb 73.

[0161] A total of 156 colonies was picked from the replicate filtercorresponding to one of the positive smaller pools, and inoculated intoculture medium for overnight growth. After overnight growth, thecultures were arranged in a matrix format of 12 rows and 13 columns.Subpools of culture medium were prepared by pooling medium from each rowand each column for a total of 24 subpools. The subpools were used toprepare DNA for a final round of transfection and screening. Anintersection of a positive row and a positive column indicated apotential positive colony. One potential positive colony (i.e. clone)was identified.

[0162] A streak plate was prepared from the positive clone (clone 1),and minipreps of DNA were made from nine individual colonies from thestreak plate. The DNA was digested with Bgl II and analyzed by SDS-PAGE.Nine of nine individual colonies from clone 1 contained identicalinserts of 1.8-2.0 Kb. A single isolate that contained the 1.8-2.0 Kbinsert was picked and inoculated into 10 ml culture medium for overnightgrowth. DNA was prepared and sequenced by dideoxynucleotide sequencing.The nucleotide and deduced amino acid sequence of clone 1 is given inSEQ ID NO:1. A cloning vector containing human CD39 sequence, designatedpCD39 was deposited with the American Type Culture Collection,Rockville, Md. (ATCC) on Sep. 29, 1992, under the Budapest Treaty, andassigned accession number 69077. A murine homolog of CD39 was isolatedby cross-species hybridization; the amino acid sequence of the murinehomolog is described in Maliszewski et al., J. Immunol. 153:3574, 1994.

Example 5 Preparation of CD39 mAbs

[0163] This example describes the preparation of additional monoclonalantibodies against CD39, including antibodies against the region thatcontains apyrase activity. Preparations of purified CD39 fragmentsexhibiting ADPase activity, for example, or transfected cells expressingsuch CD39 polypeptides, are employed as immunogens to generatemonoclonal antibodies against CD39 using conventional techniques, suchas those disclosed in U.S. Pat. No. 4,411,993. DNA encoding CD39fragments can also be used as an immunogen, for example, as reviewed byPardoll and Beckerleg in Immunity 3:165, 1995. Such antibodies areuseful for interfering with CD39-induced platelet aggregation, ascomponents of diagnostic or research assays for CD39 or CD39 activity,and in affinity purification of CD39.

[0164] To immunize rodents, CD39 immunogen is emulsified in an adjuvant(such as complete or incomplete Freund's adjuvant, alum, or anotheradjuvant, such as Ribi adjuvant R700 (Ribi, Hamilton, MT), and injectedin amounts ranging from 10-100 μg subcutaneously into a selected rodent,for example, BALB/c mice or Lewis rats. DNA may be given intradermally(Raz et al., Proc. Natl. Acad. Sci. USA 91:9519, 1994) orintramuscularly (Wang et al., Proc. Natl. Acad. Sci. USA 90:4156, 1993);saline has been found to be a suitable diluent for DNA-based antigens.Ten days to three weeks days later, the immunized animals are boostedwith additional immunogen and periodically boosted thereafter on aweekly, biweekly or every third week immunization schedule.

[0165] Serum samples are periodically taken by retro-orbital bleeding ortail-tip excision for testing by dot-blot assay (antibody sandwich),ELISA (enzyme-linked immunosorbent assay), immunoprecipitation, or othersuitable assays, including FACS analysis. Following detection of anappropriate antibody titer, positive animals are given an intravenousinjection of antigen in saline. Three to four days later, the animalsare sacrificed, splenocytes harvested, and fused to a murine myelomacell line (e.g., NS1 or preferably Ag 8.653 [ATCC CRL 1580]). Hybridomacell lines generated by this procedure are plated in multiple microtiterplates in a selective medium (for example, one containing hypoxanthine,aminopterin, and thymidine, or HAT) to inhibit proliferation ofnon-fused cells, myeloma-myeloma hybrids, and splenocyte-splenocytehybrids.

[0166] Hybridoma clones thus generated can be screened by ELISA forreactivity with CD39, for example, by adaptations of the techniquesdisclosed by Engvall et al., Immunochem. 8:871, 1971 and in U.S. Pat.No. 4,703,004. A preferred screening technique is the antibody capturetechnique described by Beckman et al., J. Immunol. 144:4212, 1990.Positive clones are then injected into the peritoneal cavities ofsyngeneic rodents to produce ascites containing high concentrations (>1mg/ml) of anti-CD39 monoclonal antibody. The resulting monoclonalantibody can be purified by ammonium sulfate precipitation followed bygel exclusion chromatography. Alternatively, affinity chromatographybased upon binding of antibody to protein A or protein G can also beused, as can affinity chromatography based upon binding to CD39polypeptide. An alternative strategy is to employ full-length CD39immunogen, selecting for antibodies that bind CD39, and winnowing outthose that bind to previously defined epitopes, for example by screeningwith a fragment of CD39 that represents a previously defined epitope.

[0167] Monoclonal antibodies are also prepared by immunizing CD39knockout mice, such as those described in Example 19D, with CD39immunogen. Since the entire CD39 sequence is seen as “foreign” in theknockout mice, this strategy can lead to the generation of antibodiesrecognizing epitopes that are shared across species lines, includingantibodies that antagonize or agonize CD39 bioactivity.

Example 6 Physiological Activity of CD39

[0168] This example demonstrates that CD39 is the endothelial cellecto-ADPase responsible for inhibition of platelet function. Humanumbilical vein endothelial cells (HUVEC) constitutively inhibit plateletresponsiveness to prothrombotic stimuli by catabolism of exogenousplatelet-derived ADP. The endothelial ecto-ADPase has been identified asCD39 (Marcus et al., J. Clin. Invest. 99:1351, 1997). Anti-CD39antibodies immunoprecipitated ADPase activity from a preparation derivedfrom endothelial membranes, and COS cells transfected with an expressionvector comprising CD39 acquired ecto-ADPase activity whereas COS cellstransfected with a control vector did not. Ecto-ADPase activity wasmeasured in a manner similar to that described in Marcus et al., J.Clin. Investigation 88:1690, 1991, by conversion of ¹⁴C-ADP to AMP bytransfectant monolayers as well as membrane preparations, and wasgreater than or comparable to activity of intact HUVEC monolayers andsolubilized membranes

[0169] HUVEC mRNA was analyzed by RT-PCR using primer pairs derived fromthe sequence of the human CD39 lymphoid cell activation antigen withemphasis on its N-terminal portion. Lymphoid CD39 cDNA was used fordirect comparison of PCR product sizes. The data demonstrated identitybetween HUVEC and lymphoid CD39 in the 4 fragments spanning the portionanalyzed (approximately 1250 of the 1850 bp of lymphoid CD39). Northernblot analyses revealed that the mRNA for CD39 in HUVEC was expressed inthe same band pattern as in MP-1 cells, from which CD39 was originallycloned.

[0170] Confocal microscopy and fluorescence activated cell sorting,using mAb73, were used to determine if HUVEC cells expressed CD39. TheFACS protocol was substantially as described in Example 1. For confocalmicroscopy, cells (human umbilical vein endothelial or transfected COS-1cells) grown on coverslip glass were washed with PBS and fixed with 3%paraformaldehyde for 30 minutes at room temperature. Auto fluorescencewas quenched by treatment with 50 mM NH₄Cl for 10 minutes. Cells werethen incubated in PBS containing 5% NGS (normal goat serum) plus 0.1%triton X-100 to block non-specific binding and to permeabilize cells.Cells were then incubated with anti-CD39 antibody at 5 μg/ml in PBScontaining 5% NGS+0.1% triton X-100 for 1 hour at room temperature.Following three washes with PBS containing 5% NGS+0.1% triton X-100cells were incubated with goat anti-mouse labeled with Texas Red(Molecular Probes) at 5 μg/ml in addition to 10 mM YOYO (MolecularProbes) for nucleic acid counter stain, for 1 hour at room temperature.Cells were washed 3 times with PBS containing 5% NGS+0.1% triton X-100and mounted in 100 mg/ml DABCO (1,4 diaxabicyclo [2.2.2] octane) (Sigma)in 50% glycerol. Cells were then viewed with Multiprobe 2001 laserscanning confocal microscope (Molecular Dynamics). One image wascollected of CD39 staining (Texas Red) and a second image was collectedof cell nuclei (YOYO).

[0171] Both the confocal microscopy and FACS experiments demonstratedthat HUVEC express CD39. The patterns of expression were similar tothose seen in cells transfected with full-length human CD39.

[0172] The physiological activity of CD39 was illustrated by the abilityof CD39-transfected COS cells to inhibit and completely reverse plateletaggregation by 10 μM ADP. CD39-transfected COS cells, as well as MP-1cells and HUVEC, metabolized this quantity of ADP to AMP within three tofour minutes and, when added to platelet rich plasma (substantially asdescribed in Marcus et al., supra), they rapidly reversed plateletaggregation. This activity occurred within the time frame of plateletadhesion to injured subendothelium, a process leading to immediate ADPrelease, recruitment of additional platelets and formation of ahemostatic plug or thrombus. This time course paralleled plateletinhibition by CD39-expressing cells, and was commensurate with theirADPase activities. The activity of ADPase/CD39 was independent offormation of other known thromboregulators, nitric acid or prostacyclin.These results demonstrate the importance of ADPase/CD39 as aphysiological, constitutively expressed endothelial cellthromboregulator.

Example 7 Phosphate Release Assay for ATPase Activity

[0173] This example describes an ATPase assay that may be used to trackenzyme activity. Samples (approximately 100 μl of either concentrated CMor purified polypeptide) are combined with 20 μl of 10X assay buffer(200 mM HEPES, 1.2 M NaCl, 50 mM KCl, 15 mM CaCl₂, 15 mM MgCl₂ and 3 mMATP) and sterile water is added to a final volume of 200 μl.Radiolabeled ATP (0.8 μCi γ [³²P] ATP; Amersham, Arlington Heights,Ill.) is added and the mixture incubated for 20 minutes at 37° C. Stopmix (0.5 ml of 20% activated charcoal/1 M HCl) is added and the reactionis placed on ice for 10 minutes. After centrifugation (14K rpm for 10minutes), the supernatant is assayed for free ³²P using a scintillationcounter. Data are expressed as raw counts or net counts, or as picomolesof ATP degraded per minute.

Example 8 Binding Assay

[0174] This example describes an assay to asses the binding of CD39polypeptides to CD39 antibodies by biospecific interaction analysis(BIA) using a biosensor, an instrument that combines a biologicalrecognition mechanism with a sensing device or transducer. An exemplarybiosensor is BIAcore^(™), from Pharmacia Biosensor AB (Uppsala, Sweden;see Fägerstam L. G., Techniques in Protein Chemistry II, ed. J. J.Villafranca, Acad. Press, NY, 1991). BIAcore^(™) uses the opticalphenomenon surface plasmon resonance (Kretschmann and Raether, Z.Naturforschung, Teil. A 23:2135, 1968) to monitor the interaction of twobiological molecules. Molecule pairs having affinity constants in therange 10⁵ to 10¹⁰ M⁻¹, and association rate constants in the range of10³ to 10⁶ M⁻¹s⁻¹, are suitable for characterization with BIAcore^(™).

[0175] The biosensor chips are coated with CD39 antibody (e.g., mAb73).The different constructs of CD39 to be assessed are then added atincreasing concentrations; the chip is regenerated between the differentconstructs, for example, by the addition of sodium hydroxide. Theresultant data can analyzed to qualitatively or quantitatively assesproduction of CD39 polypeptides. Affinity of the CD39 polypeptides forthe CD39 antibodies can also be determined. In a similar manner, othermonoclonal antibodies or polypeptides that specifically bind CD39 can beimmobilized on a biosensor chip to asses the binding of various CD39polypeptides.

Example 9 Transient Expression of Soluble CD39 Polypeptides

[0176] This example describes the preparation of constructs for thetransient expression of soluble CD39 polypeptides.

[0177] A. Reagents Used

[0178] The B73 mAb, a murine IgG1 recognizing human CD39, was kindlyprovided by Dr. Guy Delespesse (U. Montreal, Quebec, Canada). The M2 mAbrecognizing the FLAG^(®) peptide (DYKDDDDK, SEQ ID NO:10), a murineIgG1, was prepared at Immunex Corp. Affigel 10 (Bio-Rad, Hercules,Calif.) and CNBr-activated Sepharose 4B (Pharmacia Biotech, Piscataway,N.J.) immunoaffinity columns were prepared according to manufacturers′instructions. Typically, coupling efficiencies in the range of 3-5 mgmAb per ml of affinity gel slurry were obtained.

[0179] B. Construction of a Soluble CD39 (solCD39) Expression Plasmid

[0180] To generate a soluble molecule having the properties of CD39 theN-terminal and C-terminal portions of CD39, including the twotransmembrane regions (see FIG. 2), were removed. To allow transport ofsoluble CD39 into the medium, a leader sequence providing for secretionwas added at the amino terminus of the polypeptide.

[0181] Constructs of soluble human CD39 (solCD39) were made in themammalian expression vector pDC206 (Kozlosky et al. Oncogene. 10:299,1995), utilizing human IL2 (huIL2), human growth hormone (huGH) andmurine IL7 (muIL7) leaders).

[0182] The DNA sequences between the putative transmembrane regions offull-length CD39, including nucleotides 178-1494 of SEQ ID NO:1, wereamplified using PCR and the C-terminal transmembrane coding region wasreplaced with a stop codon. The PCR product was fused to a synthetic DNAfragment encoding an 8 amino acid peptide tag (FLAG^(®)) and ligatedwith a muIL7 leader (muIL7L) into the plasmid pDC206 vector via Spe1 andBgl2 restriction sites. This construct encoded N-terminally FLAG-taggedsolCD39.

[0183] Alternate leaders were introduced by ligating the Spel/Bgl2FLAG-solCD39 fragment into two different pDC206 plasmids, with leadersderived from: (1) human growth hormone (huGHL), and (2) a humanproinsulin/IL2 fusion polypeptide (huIL2L, Cullen, DNA. 7:645, 1988).The coding region of the latter construct, which is shown in SEQ IDNOs:25 and 26, includes sequences encoding the huIL2 leader (huIL2L,nucleotides 1-72, amino acids 1-24 in SEQ ID NO:25), the first 12 aminoacids of mature human IL2 (nucleotides 73-108, amino acids 25-36 in SEQID NO:25), a four amino acid linker (nucleotides 109-120, amino acids37-40 in SEQ ID NO:25), the FLAG tag (nucleotides 121-144, amino acids41-48 of SEQ ID NO:25), and sol CD39 (nucleotides 145-1461, amino acids49-487 of SEQ ID NO:25).

[0184] The constructs comprising the muIL7 leader, the human growthhormone leader, and the human proinsulin/IL2 leader were designatedpIL7LFlagSolCD39, pGHLFlagSolCD39, and pIL2LFlagSolCD39 respectively.

[0185] Each construct was used to transiently transfect subconfluentlayers of COS-1 cells using DEAE dextran followed by chloroquine asdescribed by Cosman et al., Nature 312:768, 1984. As a negative control,a CD40 ligand construct (pIl2LCD40lig, Spriggs et al., J. Exp. Med.176:1543, 1992) was also transfected into COS-1 cells.

[0186] C. Preparation of Conditioned Medium from solCD39 Transfectants

[0187] The transfected COS-1 cells were incubated (37° C., 5% CO₂) in0.5% FCS-supplemented DMEM-F12 medium in 10 cm² Petri dishes or 175 cm²tissue culture flasks. After 5 days, conditioned medium (CM) from thesecultures was collected, and cells and debris were removed bycentrifugation. The CM was concentrated 4-10 fold using a pressurized,stirred cell fitted with a YM-10 (10 kD cutoff) membrane (Amicon Corp.,Danvers, Me.).

[0188] D. ATPase Activity in Conditioned Medium from solCD39Transfectants

[0189] ATPase activity in the CM from solCD39 transfectants (100 μL of10-fold concentrated supernatant) was assayed essentially as describedin Example 7, except that the 10X assay buffer contained 30 mM cold ATP.The results are shown in TABLE 1.

[0190] The transfections were repeated and the CM (10, 20 and 30 μL,unconcentrated) was assayed essentially as described in Example 7. Theresults are shown in TABLE 2. Because the pIL2LFlagSolCD39 showed higherATPase activity in COS-1 supernatants than pIL7LFlagSolCD39 andpGHLFlagSolCD39, this construct was selected for further investigation.ATPase levels in CM from COS-1 cells transfected with pIL2LFlagSolCD39increased with time in culture over at least 4 days post-transfection.TABLE 1 ATPase Activity in Concentrated CM from solCD39 TransfectantsSample CPM Release × 10³ (raw counts) pIL2LFlagSolCD39 99.96pIL7LFlagSolCD39 39.47 pGHLFlagSolCD39 21.14 pIL2LCD40lig 10.53 mediaonly 7.89

[0191] TABLE 2 ATPase Activity in CM from solCD39 Transfectants SampleVol (μL) CPM Release × 10³ (net counts) pIL2LFlagSolCD39 10 24.71 2043.92 30 56.93 pIL7LFlagSolCD39 10 5.01 20 9.75 30 14.23 pGHLFlagSolCD3910 5.51 20 7.22 30 9.95

[0192] E. Immunoaffinity Depletion of solCD39 from COS-1 CM

[0193] To confirm that recombinant solCD39 accounted for the ATPaseactivity observed in the CM, CM from COS-1 transfectants was incubatedwith immunoaffinity beads prior to enzyme assay.

[0194] CM was collected from COS-1 cells transfected withpIL2LFlagSolCD39, which had been cultured for 5 days in DMEM/F12supplemented with 5% FCS. A 100 μl aliquot of drained Affigel beads (AG)conjugated with either chicken ovalbumin, antiFLAG mAb, or anti-CD39 mAbwas added per ml of CM. CM was subjected to one or two cycles of bindingwith one of the following: ovalbumin-conjugated AG, M2 mAb-conjugatedAG, or B73 mAb-conjugated AG. Each cycle involved continuous gentleagitation of the slurry for 14 h at 4° C. followed by centrifugation torecover supernatants for a subsequent binding cycle or for ATPDaseactivity measurements.

[0195] As shown in FIG. 3, immunoprecipitation with anti-CD39mAb-conjugated beads resulted in removal of over 80% of ATPase activityfrom CM. Over 95% ATPase activity was removed with a second antibodyadsorption step. Immunoprecipitation (2 cycles) with anti-FLAGmAb-coated beads also resulted in substantial depletion of enzymeactivity. Two rounds of preincubation with a control(ovalbumin-conjugated beads) did not remove significant ATPase activityfrom the supernatants.

[0196] F. Immunoprecipitation of Recombinant solCD39

[0197] To characterize recombinant solCD39 polypeptide expression, COS-1cells were transfected with mammalian expression vectors encoding cellsurface CD39 (pHuCD39, Marcus et al., J. Clin. Invest. 99:1351, 1997),tagged soluble CD39 (pIL2LFlagSolCD39), or soluble CD40 ligand(pIL-2L-CD40L) and grown in 5% FCS-supplemented DMEM/F12 medium in 10cm² dishes. Two days after transfection, the medium was replaced withCys/Met-free medium and cells were incubated for 1 h at 37° C. Theculture medium was replaced with fresh Cys/Met-free medium supplementedwith 5 μl of [³⁵S]-Cys/Met (Amersham, Arlington Heights, Ill.) in orderto label newly synthesized polypeptides, and cells were cultured for 5 hat 37° C. CM from the metabolically radiolabeled cells was collected,purified of cells and debris by centrifugation and sterile filtration,and stored at 4° C. until further use.

[0198] For radioimmunoprecipitation, 500 μl of ³⁵S-labeled CM was addedto 250 μl of 3% BSA in Tris-buffered saline (TBS), pH 7.7, followed byaddition of 50 μl of a 80% slurry of mAb-coated AG beads. In some cases,³⁵S-labeled CM were incubated with ovalbumin-coated AG beads to removenonspecifically binding materials prior to addition of Ab-coated beads.After incubation for 14 h at 4° C., beads were removed by centrifugationand washed three times in cold TBS.

[0199] For SDS-PAGE analysis, 35 μl of 4-fold concentrated reducingsample buffer (250 mM Tris/HCl, pH 6.8, 8% (w/v) SDS, 40% (v/v)glycerol, 20% (v/v) 2-mercaptoethanol, 0.05% bromophenol blue dye) wasadded to each AG pellet, boiled for 5 min, and loaded onto a 8-16% Novex(San Diego, Calif.) polyacrylamide gel. Gels were electrophoresed at 25mA, prepared for autoradiography by soaking in Enhance (NEN Life ScienceProducts, Boston, Mass.) for 1 h and in H₂O for 20 min, followed byvacuum drying at 80° C. Gels were exposed to Kodak (Rochester, N.Y.)X-omat AR film for 2 h, then developed.

[0200] As shown in FIG. 4, IL-2L-FlagsolCD39 transfectants secreted aradiolabeled protein of ˜66 kD that was recognized by anti-CD39. Thisprotein was not detected in anti-CD39 immunoprecipitated CM from COS-1cells transfected with a vector encoding full-length CD39 (includingN-terminal and C-terminal hydrophobic regions), or with a vectorencoding a secreted protein, CD40 ligand. Anti-FLAG mAbimmunoprecipitated a similar-sized band from CM of the pIL-2LFlagsolCD39 transfectant, consistent with the presence of the FLAG^(®)peptide in recombinant solCD39. Preclearing radiolabeled culturesupernatants with anti-ovalbumin-coated beads failed to remove the 66 kDband, indicating that binding to anti-CD39 and anti-FLAG was specific.Beads coated with an irrelevant, isotype matched control antibody failedto immunoprecipitate the 66 kD band from solCD39 containing CM.

[0201] G. Preparation of Additional solCD39 Fusion Constructs

[0202] Restriction enzymes were used to prepare a DNA fragmentcomprising nucleotides 1 through 1488 of SEQ ID NO:1, the coding regionof which would be expected to encode a fragment of CD39 lacking thesecond (most C-terminal) transmembrane domain. Appropriate linkeroligonucleotides were prepared (SEQ ID NOs:14 and 15), and used in athree-way ligation of the CD39 DNA, the linker oligonucleotides, and anexpression vector comprising regulatory elements allowing expression ofa resulting fusion polypeptide in mammalian cells along with DNAencoding a mutated human immunoglobulin Fc (SEQ ID NOs:16 and 17) regionthat exhibits reduced affinity for Fc receptors (nucleotides 42 through740 of SEQ ID NO:16). This construct was referred to as CD39Δ2Fc, andwhen transfected into cells resulted in the expression of a fusionpolypeptide comprising amino acids 1 through 474 of SEQ ID NO:1 andamino acids 1 though 232 of SEQ ID NO:17, which could be detected on thesurface of transfected cells using either anti-CD39 or anti-human IgG.

[0203] A PCR technique was employed to prepare a fragment of DNA fromthe CD39Δ2Fc construct that also lacked the first, most amino-proximaltransmembrane region, but included CD39 DNA from nucleotides 181 through1488 (amino acids 39 through 474) of SEQ ID NO:1 and the Fc mutein DNAfrom CD39Δ2Fc (using the linkers shown in SEQ ID NOs:18 and 19).

[0204] The resulting DNA was then ligated into an expression vector thatincluded DNA encoding the murine Interleukin-7 leader sequence (SEQ IDNO:20) ligated immediately proximal to the CD39-encoding sequence. Thisconstruct was designated CD39Δ1,Δ2Fc.

[0205] DNA encoding the FLAG^(®) peptide (SEQ ID NO:10) and a codoncorresponding to nucleotides 178, 179 and 180 of SEQ ID NO:1 wasinserted into the CD39Δ1,Δ2Fc construct, in between the leader sequenceand the CD39-encoding sequence, to provide a detectable tag for theamino terminus of the fusion polypeptide (using the linkers shown in SEQID Nos:21 and 22). The tagged construct was referred to asFlagCD39Δ1,Δ2Fc.

[0206] Another construct was prepared that removed the Fc muteinsequences, and added codons corresponding to nucleotides 1489 through1494 of SEQ ID NO:1 immediately downstream of the CD39 sequences (usingthe linkers shown in SEQ ID Nos:23 and 24). This construct wasdesignated FlagCD39Δ1,Δ2.

[0207] Each of the constructs was transfected into mammalian cells andprotein levels were assayed on the surface, in the interior, or in thesupernatant fluid of transfected cells using antibodies to FLAG^(®),CD39, or human IgG.

Example 10 Expression and Activity of pIL2LSolCD39

[0208] To facilitate the establishment of a stably producingtransfectant in CHO cells, an untagged version of soluble human CD39(solCD39) was constructed. A 523 bp Spel/Ndel fragment containing theFLAG^(®) tag and the first 163 amino acids (aa) of pIL2LFlagSolCD39 wasremoved, and replaced with a similar fragment from a C-terminallyFLAG^(®)-tagged solCD39. Thus the entire solCD39 coding region wasreconstituted, sans FLAG^(®), while retaining the HuIL2 leader andmature IL2 residues. This construct was designated pIL2LSolCD39. Thecoding region of pIL2LSolCD39, which is shown in SEQ ID NO:7, includessequences encoding the huIL2 leader (huIL2L, nucleotides 1-72, aminoacids 1-24 in SEQ ID NO:7), the first 12 amino acids of mature human IL2(nucleotides 73-108, amino acids 25-36 in SEQ ID NO:7), a three aminoacid linker (nucleotides 109-117, amino acids 37-39 in SEQ ID NO:7), andsol CD39 (nucleotides 118-1434, amino acids 40-478 of SEQ ID NO:7).

[0209] To determine whether activity was affected by removal of theN-terminal FLAG^(®) tag, COS-1 cells were transfected withpIL2LFlagSolCD39 and pIL2LSolCD39 and supernatants (sups) were harvestedafter 5 days. Samples of 10, 20 and 30 μL of 1x sups were assayed forATPase activity as described in Example 7. As shown in TABLE 3, activitywas not affected by removal of the N-terminal FLAG^(®) tag. TABLE 3ATPase Activity in CM from pIL2LFlagSolCD39 and pIL2LSolCD39Transfectants Sample Vol (μL) CPM × 10³ (net counts) pIL2LFlagSolCD39 1023.6 20 40.2 30 54.4 pIL2LSolCD39 10 20.1 20 36.0 30 51.0

Example 11 Preparation of Additional solCD39 Fusion Constructs

[0210] A. Preparation and Characterization of Trim1 and Trim2 Variants

[0211] To characterize the effect of the 12 mature human IL2 (huIL2)residues on the expression of solCD39, the huIL2 residues were removedduring the construction of nucleic acid sequences encoding twoadditional variants of pIL2LSolCD39: pIL2LTrim1 (“Trim1”) and pIL2LTrim2(“Trim2”).

[0212] The pIL2LTrim1 variant was constructed by purifying a Hind3/Bgl2restriction fragment from pIL2LSolCD39 which contained the entiresolCD39 coding region except for the first four amino acids. Thisfragment was ligated with a synthetic oligo cassette (containing thehuIL2 leader and the first amino acid of mature huIL2) into Sma1/Bgl2digested pDC206. The huIL2 leader was thus reintroduced and joined tosolCD39 with an intervening alanine residue.

[0213] The pIL2LTrim2 variant was constructed in a similar fashion usinga Spe1/Bgl2 fragment from pIL2LSolCD39 and a synthetic oligo cassettecontaining the huIL2 leader and the linker-encoded sequences (with thefirst codon altered to alanine). Thus, the huIL2 leader was restoredwith an intervening Ala-Ser-Ser linker preceding solCD39.

[0214] The N-terminal portions of the pIL2LSolCD39, pIL2LTrim1 andpIL2LTrim2 polypeptides are compared below, with the predicted cleavagepoints indicated as *:

[0215] pIL2LSolCD39 (SEQ ID NO:11)MALWIDRMQLLSCIALSLALVTNS*APTSSSTKKTQLts sT QNK . . .

[0216] pIL2LTrim1 (SEQ ID NO:12) MALWIDRMQLLSCIALSLALVTNS A T*QNK . . .

[0217] pIL2LTrim2 (SEQ ID NO:13) MALWIDRMQLLSCIALSLALVTNS as*sT QNK . ..

[0218] The polypeptide encoded by the Trim1 construct has the sequenceSEQ ID NO:27. Residues 26-464 are a soluble portion of CD39 and thecleavage of the leader sequence is between Ser24 and Ala25.

[0219] The expression of Trim1 and Trim2 constructs was analyzed inCOS-1 cells cultured in 10 cm plates. After 5 days of incubation, 1xsupernatants were examined via ELISA (using anti-CD39) and thephosphate-release assay described in Example 7. As shown in TABLE 4, thespecific activities (based on concentrations determined by ELISA) ofTrim1 and Trim2 were equivalent to pIL2LFlagSolCD39. Expression levels,however, appeared to be reduced 3-4 fold. TABLE 4 SolCD39 Expression andActivity in CM from pIL2LSolCD39 an Trim Transfectants Sample [CD39]μg/mL Activity (pmol ATP/min/μg) × 10³ pIL2LSolCD39 0.75 5.67 pIL2LTrim10.21 8.38 pIL2LTrim2 0.21 6.67

[0220] COS-1 cells were also transfected with pIL2LSolCD39, pIL2LTrim1and pIL2LTrim2 and cultured in T175 flasks (30 mL). 5-day/1x sups werethen analyzed via ELISA. As shown in TABLE 5, the ELISA results againindicated lower expression levels for the Trim1 and Trim2 variants.

[0221] To further characterize the effect of the human IL2 (huIL2)residues on the expression of solCD39, the pIL2LSolCD39, pIL2LTrim1, andpIL2LTrim2 products were purified using anti-CD39 coated sepharose. TheN-terminal amino acid sequence was determined for each of the purifiedpolypeptides. For solCD39 the N-terminus was APTSSSTKKT . . . (residues25-34 of SEQ ID NO:11). For Trim1 the N-terminus was ATQNKALPEN . . .(residues 25-34 of SEQ ID NO:27). The Trim2 polypeptides hadheterogeneous N-termini. TABLE 5 SolCD39 Expression in CM frompIL2LSolCD39 and Trim Transfectants Sample [CD39] μg/mL pIL2LSolCD390.796 pIL2LTrim1 0.143 pIL2LTrim2 0.113

[0222] B. Preparation and Characterization of Trim3 and Trim4 Variants

[0223] Nucleic acids encoding additional solCD39 variants, designatedpIL2LTrim3 (“Trim3”) and pIL2LTrim4 (“Trim4”), are also constructedusing a synthetic oligo cassette strategy. The N-terminal portions ofthe solCD39, Trim3 and Trim4 polypeptides are compared below. Thepredicted cleavage points are indicated as *.

[0224] pIL2LSolCD39 MALWIDRMQLLSCIALSLALVTNS*APTSSST KKTQLtssTQNK . . .(SEQ ID NO:11)

[0225] pIL2LTrim3 MALWIDRMQLLSCIALSLALVTNS*A ST KKTQLtssTQNK . . . (SEQID NO:28)

[0226] pIL2LTrim4 MALWIDRMQLLSCIALSLALVTNS ST*KKTQLtssTQNK . . . (SEQ IDNO:29)

[0227] The polypeptide encoded by the Trim3 construct has the sequenceSEQ ID NO:28. Residues 36-474 are a soluble portion of CD39 and thepredicted cleavage of the leader sequence is between Ser24 and Ala25.The polypeptide encoded by the Trim4 construct has the sequence SEQ IDNO:29. Residues 35-473 are a soluble portion of CD39 and the predictedcleavage of the leader sequence is between Thr26 and Lys27.

[0228] The pIL2LTrim3, and pIL2LTrim4 polypeptides are expressed andpurified using anti-CD39 coated sepharose. The N-terminal amino acidsequence and specific activity are determined for each of thepolypeptides.

[0229] C. Preparation and Characterization of solCD39-L4 FusionPolypeptides

[0230] The CD39 gene family is reported to contain at least four humanmembers: CD39, CD39L2, CD39L3, and CD39L4 (Chadwick and Frischauf,Genomics 50:357, 1998). CD39-L4 is reported to be a secreted apyrase(Mulero et al., J. Biol. Chem. 274(29):20064, 1999). Additional solCD39variants are constructed by fusing N-terminal sequences from CD39L2,CD39L3, or CD39-L4 to a soluble portion of CD39. The N-terminal aminoacid sequences of human CD39 and human CD39-L4 are aligned in FIG. 24.

[0231] For one construct, CD39-L4-1, a nucleic acid encoding CD39-L4amino acid residues 1-37 (Met1 to Ser37 of SEQ ID NO:31) is fused to anucleic acid encoding CD39 residues 38-476 (Thr38 to Thr476 of SEQ IDNO:2). The polypeptide encoded by the CD39-L4-1 construct has thesequence SEQ ID NO:3. Residues 1-37 are from CD39-L4, residues 38-476are a soluble portion of CD39, and the predicted site of cleavage of theleader sequence is between Ala20 and Val21.

[0232] For another construct, CD39-L4-2, a nucleic acid encoding CD39-L4amino acid residues 1-48 (Met1 to Leu48 of SEQ ID NO:31) is fused to anucleic acid encoding CD39 residues 49-476 (Tyr49 to Thr476 of SEQ IDNO:2). Another construct, CD39-L4-3, is identical to CD39-L4-2 exceptthat the Cys residue at position 39 (Cys39) is replaced by another aminoacid, preferably Ser. The polypeptides encoded by the CD39-L4-2 andCD39-L4-3 constructs have the sequence SEQ ID NO:4. Residues 1-48 arefrom CD39-L4, residues 49-476 are a soluble portion of CD39, and thepredicted site of cleavage of the leader sequence is between Ala20 andVal21.

[0233] Additional constructs are constructed by fusing a portion of theCD39-L4 N-terminal coding region to the CD39 N-terminal coding region.After expression in recombinant cells, the N-terminal sequence,enzymatic activity, and platelet inhibitory activity is determined foreach of the polypeptide products.

[0234] D. Preparation and Characterization of IgkappaLsolCD39

[0235] Nucleic acids encoding an Igkappa leader sequence fused to aminoacids from IL-2 and to solCD39 are also constructed. One such constructencodes a polypeptide having an Igkappa leader and four amino acids fromIL-2 fused to the N-terminus of the CD39 soluble portion (set forth asresidues Thr38 to Thr476 of SEQ ID NO:2). The N-terminus of the encodedpolypeptide is therefore: 5′-METDTLLLWVLLLWVPGSTG*APTSTQNKALPE . . .(amino acids 1-32 of SEQ ID NO:30), where amino acids Met1-Gly20 are theIgkappa leader, Ala21-Ser24 are from IL-2, and Thr25-Glu32 is thebeginning of solCD39 sequences. The predicted cleavage site is indicatedas *. The polypeptide encoded by the IgkappaLsolCD39 construct has thesequence SEQ ID NO:30. Residues 25-463 are a soluble portion of CD39 andthe predicted cleavage of the leader sequence is between Gly20 andAla21. After expression in recombinant cells, the N-terminal sequence,enzymatic activity, and platelet inhibitory activity is determined foreach polypeptide product.

Example 12 Development of a Stably Transfected Cell Line SecretingsolCD39

[0236] A CHO cell line expressing solCD39 was generated to improverecombinant solCD39 polypeptide production.

[0237] A. Preparation of Constructs and Cell Lines for the StableExpression of Soluble CD39 Polypeptides

[0238] The solCD39 cDNA insert, containing the recombinant solCD39sequence and the IL-2 leader but not the FLAG^(®) sequence, was excisedfrom the pIL2LSolCD39 plasmid by XmaI/BglII digestion, then insertedinto 2A5Ib, an expression vector containing the DHFR gene and optimizedfor stable CHO cell lines (Morris et al., In Animal Cell Technology:From Vaccines to Genetic Medicine, M. J. T. Carrondo, B. Griffiths, andJ. L. P. Moreira, editors, Kluwer Academic Publishers, Boston. 529-534,1997).

[0239] The solCD39-2A5Ib plasmid was transfected into CHO cells usingLipofectamine (GIBCO BRL; Gaithersburg, Md.) according to manufacturer'srecommendations. The CHO cell line used in these studies, DX B-11, hadbeen adapted to serum-free suspension culture conditions. Transfectedcells were grown in modified DMEM-F12 medium, supplemented with peptone,glutamine, glucose, transferrin, lipids, and IGF-1 (insulin-like growthfactor 1; used solely when cultures were induced for proteinexpression). After 3 days growth, the cells were transferred toselective medium lacking hypoxanthine and thymidine. Stock cultures weregrown at 37° C. in suspension, and passaged every 2-3 days. Inductioncultures were grown at 31° C. in suspension, with IGF-1 and sodiumbutyrate (1-2 mM). Cell density at start of induction cultures was1.5-2×10⁶ cells/ml. The average induction period was 7 days, at whichtime CM was collected for further analyses.

[0240] B. TLC Assays for ADPase and ATPase Activities in CM ContainingsolCD39

[0241] Following growth in selective medium, CM from CHO cell cultureswas analyzed for ATPase and ADPase activities. ADPase assays (Marcus etal., J. Clin. Invest. 88:1690, 1991) were primarily used in determiningenzyme kinetics and pharmacokinetics. Test samples were incubated with50 μM [⁴C] ADP (NEN Life Science Products) in assay buffer (15 mM Tris,134 mM NaCl, 5 mM glucose, pH 7.4, containing 10 μM Ap5A(P¹,P⁵-di[adenosine-5′]pentaphosphate, 1 mM ouabain, 10 μM dipyridamole,and 3 mM CaCl₂) in a total volume of 50 μl (5 min, 37° C.). Reactionswere stopped by placement on ice and addition of 10 μl “stop solution”(160 mM disodium EDTA, pH 7.0, 17 mM ADP, 0.15 M NaCl) to block furthermetabolism of ADP. Nucleotides, nucleosides, and bases were separated byTLC using isobutanol/1-pentanol/ethylene glycol monoethylether/NH₄OH/H₂O (90: 60: 180: 90: 120). Radioactivity was quantified byradio TLC scanning (RTLC multiscanner; Packard, Meriden, Conn.). Resultswere calculated as averages of duplicate to quadruplicate measurementsafter subtraction of buffer blanks (consistently <1% of totalradioactivity). Data were expressed as percentage of ADP metabolized oras pmol ADP metabolized per minute per μl CM. A unit of activity is thequantity of enzyme which will degrade 1 μmol of ADP in 1 min at 37° C.Identical assays were performed using ATP as a substrate in order toexamine the kinetics of the ATPase activity of CD39.

[0242] As shown in TABLE 6, the stably transfected CHO cells secreted20-fold higher levels of both enzyme activities compared to CM fromtransfected COS-1 cells. TABLE 6 Comparison of ADPase and ATPaseActivities in CM Containing solCD39 Cell Type ADPase (pmol/min/μl)ATPase (pmol/min/μl) CD39 (CHO) 1403 970 CD39 (COS-1)  70  44

[0243] C. Affinity Purification of solCD39 from Stably Transfected CHOCells

[0244] Thirty ml of 10-fold concentrated CM from solCD39-secreting CHOcells was added to 3 ml of B73 mAb-coated Sepharose 4B gel slurry andmixed overnight at 4° C. The affinity matrix was pelleted bycentrifugation, washed 3 times with PBS, and added to a plastic column.Specifically-bound protein was eluted by the addition of 0.1 Mtriethylamine, pH 11.5. Fractions (1.2 ml each) were collected in tubescontaining 120 μl of neutralizing solution (1 M sodium phosphate,monobasic; pH 4.3) and analyzed for protein content by SDS-PAGE,followed by Coomassie Blue staining. Biological activity was determinedusing an ATPase assay as described in Example 7, so that peak fractionscould be pooled, buffer exchanged into PBS, and concentrated 5-fold.N-linked sugars were removed from purified protein using a kit fromOxford Glycosystems (Rosedale, N.Y.). The recombinant solCD39 wasanalyzed by SDS-PAGE.

[0245] A prominent band of ˜66 kD was present in early eluted fractions,with a peak of Coomassie Blue staining around fraction 5 (FIG. 5A). Over90% of the protein present was found as this major band. Overloading thepolyacrylamide gel did reveal some smaller molecular weightcontaminants, however, these appear to be related to the antibodiespresent on the column and not to the protein loaded on the column.

[0246] ATPDase activity of the affinity column fractions correlated withthe intensity of protein bands on SDS-PAGE (FIG. 5A, 5B). ATPDaseactivity was barely detectable in the anti-CD39 column flowthrough,indicating that affinity purification is an effective means of isolatingbiologically active recombinant solCD39. Treatment of the purifiedprotein with N-glycanase for 18 hours to remove N-linkedoligosaccharides caused the broad band of protein at 66 kD to resolveinto a much tighter band of protein at approximately 52 kD, thepredicted size for solCD39 (FIG. 5C).

[0247] The total protein yield from 1 L of CHO-solCD39 CM was ˜2 mg.Production of solCD39 was scaled up to 10 liter bioreactors. Theresultant conditioned medium contained approximately 50-100 μg/ml ofsolCD39 according to ELISA analysis. Thus, each 10 L bioreactor runwould expected to produce 500-1000 mg of recombinant polypeptide. CHOcell lines expressing additional solCD39 constructs are similarlyprepared and characterized.

Example 13 Expression of solCD39 in Veggie-CHO and CS-9 Cells

[0248] In this example, soluble CD39 is expressed in CHO cells that havebeen adapted to grow in suspension in media that does not contain animalproteins (see Rasmussen et al., Cytotechnology 28:31, 1998), or in thepresence of IGF-1 in the clonal cell line CS-9.

[0249] The dihydrofolate reductase-deficient Chinese hamster ovary cellline, DXB11-CHO is commonly used as a host cell for the production ofrecombinant polypeptides. DXB11-CHO was adapted to grow in suspension. Aserum-free host named Veggie-CHO was then generated by adaptingDXB11-CHO cells to growth in serum-free media in the absence ofexogenous growth factors such as Transferrin and Insulin-like growthfactor (IGF-1). The latter adaptation was achieved by a gradualreduction of serum supplementation in the media and the replacement ofserum with low levels of growth factors, IGF-1 and transferrin, in anenriched cell growth media. The suspension adapted serum-free adaptedcells were then weaned off these growth factors. The resultingVeggie-CHO cells maintain vigorous growth and high viability as well asa DHFR-deficient phenotype in media that is serum-free and also free ofanimal-derived proteins. CS-9 cells were also derived from DXB11-CHOcells. The suspension adapted serum-free adapted cells were adapted togrow in the absence of transferrin, then individual clones wereisolated. The CS-9 clone was chosen for its stable recombinant proteinexpression.

[0250] Veggie-CHO cells and CS-9 cells are used as a host cell line forthe stable, high level expression soluble CD39 polypeptides usingmethods similar to those described in Example 12.

Example 14 Biochemical Properties of Affinity-Purified solCD39

[0251] Purified solCD39 material was subjected to N-terminal amino acidsequencing and mass spectroscopy. Quantitative amino acid analysis ofpeak fractions (3-9) from the affinity column yielded a ratio of aminoacid residues consistent with calculated values for human CD39. TheN-terminus of the pIL2LSolCD39 product had the following sequence:

[0252] APTSSSTKKTQLtssTQ . . . (residues 25-41 of SEQ ID NO:11).

[0253] The first 12 residues represent the mature huIL2 residues;residues 13-15 (tss, lower case) are linker-encoded residues; residues16,17, etc. (T Q . . . ) are solCD39.

[0254] Using the TLC assay system described in Example 12B, the ADPaseactivity of the membrane-bound HUVEC ecto-ADPase was determined atdifferent pHs in buffers containing 100 mM bis-trispropane (Sigma, St.Louis, Mont.). This was compared to the ADPase activity of purifiedsolCD39 at these pHs. Kinetic constants for CD39 metabolism of ATP andADP were determined by measuring the initial rates of reaction asanalyzed in the TLC system. ADP or ATP at 2.5-150 μM were incubatedseparately with 2×10⁻⁹ M solCD39.

[0255] As shown in FIG. 6A, the pH optima for the ecto-ADPase on thesurface of HUVEC and for affinity-purified recombinant solCD39 ADPaseactivities were between pH 8 and 8.5. This indicated that recombinantsolCD39 would be maximally active under the same physiologicalconditions as native CD39/ecto-ADPase.

[0256] Initial rates of ATP and ADP metabolism by recombinant solCD39were determined as shown in FIG. 6B, and kinetic constants were derived.The K_(m) and V_(max) for ADP were 5.9 μM and 72 pmol/min, respectively;for ATP a K_(m) of 2.1 μM and V_(max) of 26 pmol/min were determined.The assays were performed with 2×10⁻⁹ M solCD39, yielding k_(cat) of 720min⁻¹ (ADP) and 260 min⁻¹ (ATP). Thus, the specificity constant,k_(cat)/K_(m) (1.2×10 ⁸ min⁻¹ M⁻¹), was identical for ADP and ATP. Thespecific activity for purified recombinant solCD39 was 11 U/mg for ADPand 4 U/mg for ATP.

Example 15 Platelet Inhibitory Properties of solCD39

[0257] This example shows that recombinant affinity purified solCD39 iseffective as an inhibitor of platelet activation and recruitment.

[0258] After obtaining informed consent from volunteers, blood wascollected via plastic tubing using acid citrate-dextrose (38 mM citricacid; 75 mM sodium citrate; 135 mM glucose) as anticoagulant. Whereindicated, volunteers had ingested 650 mg acetylsalicylic acid (ASA) 18h prior to blood donation. Platelet-rich plasma (PRP) was prepared withan initial whole blood centrifugation (200 g, 15 min, 25° C.), and asecond centrifugation of the PRP (90 g, 10 min) to eliminate residualerythrocytes and leukocytes. The stock suspension of PRP was maintainedat room temperature under 5% CO₂-air.

[0259] A. Platelet Aggregation Studies

[0260] PRP containing 1.22×10⁸ platelets was pre-incubated (3 min, 37°C.) in an aggregometer cuvette (Lumiaggregometer; Chrono-Log, Havertown,Pa.) alone or in combination with test samples containing solCD39. Totalvolumes were adjusted to 300 μl with TSG buffer (Marcus et al., J. Clin.Invest. 88:1690, 1991; Marcus et al., J. Clin. Invest. 99:1351, 1997).After the 3 min preincubation, platelet agonists (ADP or collagen) wereadded at the concentrations indicated, and the aggregation responserecorded for 4-5 min. Where indicated, 10 μM indomethacin (Sigma, St.Louis, Mont.) was added to PRP to inhibit endogenous cyclooxygenaseactivity.

[0261] As shown in FIG. 7, the addition of 10 μM ADP to PRP aloneresulted in a full, irreversible aggregation response; partiallyreversible aggregation occurred at lower ADP concentrations. However, inthe presence of only 3.3 μg/ml solCD39, platelet aggregation induced by10 μM ADP was abruptly terminated and the curve rapidly returned tobaseline. Importantly, the extent of aggregation was reduced to levelsbelow those observed with 1 μM ADP. Higher concentrations of solCD39 hadan even more profound inhibitory effect, virtually eliminating theinitial burst of aggregation elicited by 10 μM ADP.

[0262] Platelet responsiveness to 5 μM ADP was examined in PRP treatedwith and without the cyclooxygenase inhibitor indomethacin (10 μM), inthe presence of CM containing solCD39 from COS-1 and CHO cells. As shownin FIG. 8A, indomethacin treatment resulted in partial reversal ofADP-induced platelet aggregation in the absence of solCD39. In contrast,CM containing solCD39 were capable of completely abrogating plateletresponses to ADP, whether PRP was indomethacin-treated or not.

[0263] Inhibition of platelet reactivity by CD39 was not limited toblocking the agonistic effects of ADP, as shown in FIG. 8B and 8C.Collagen, which is another critical platelet agonist, was used at 1μg/ml to induce platelet aggregation. The presence of solCD39 markedlyreduced the response to collagen compared to control (FIG. 8B, uppercurves). A similar inhibitory effect of solCD39 was observed in PRPtreated with indomethacin (FIG. 8B, lower curves), when collagen wasused at 3.3 μg/ml. As shown in FIG. 8C, the effect of solCD39 oncollagen-induced aggregation was dose dependent.

[0264] B. Inactivation of Enzymatic Activity of solCD39 and the Effecton Inhibition of Platelet Activation

[0265] To demonstrate that the ability of solCD39 to inhibit plateletactivation and recruitment was due to the enzymatic activity of solCD39and not to some other property, the solCD39 was reacted with FSBA(Fluorosulfonylbenzoyl-adenosine), an ATP analog that inhibitscollagen-induced platelet activation (Colman et al., Blood 68:565, 1986)and binds irreversibly with ATPDases found on several cell types(Sevigny et al., Biochem. Biophys. Acta 1334:73, 1997; Sevigny et al.,Biochem. J., 312:351, 1995).

[0266] SolCD39 (2 nmol) was combined with 2 ml labeling buffer (100 mMHepes, pH 7.4, 200 mM NaCl, 4% dimethylformamide [vol/vol]), 400 μl 5 mMFSBA (Sigma Chemical Co.) dissolved in ethanol, and 1.52 ml water. Amock-treated sample was also prepared in which the FSBA solution wasreplaced with water. After incubating at 37° for 90 min., the sampleswere centrifuged in a Centricon-10 filter unit (Amicon Corp.) for 1 hourat 5,500 rpm and buffer exchanged into PBS to remove unreacted material.The effect of FSBA-treated solCD39 on platelet reactivity is shown inFIG. 9.

[0267] Induction of platelet activation by ADP (FIG. 9A) or collagen(FIG. 9B) was significantly inhibited by either purified solCD39 or mockFSBA-treated solCD39. In contrast, incubation with FSBA-treated solCD39did not have a significant effect on platelet activation. A comparativetitration of mock-treated solCD39 verses FSBA-treated solCD39 (FIG. 9C)indicated that 22.0 μg/ml of FSBA-treated sol CD39 gave a similaraggregation profile as 0.88 μg/ml of mock-treated solCD39. Thisindicated that 96% of the aggregation inhibitory activity of solCD39 waslost after FSBA derivitization. Analyses of residual ADPase activity ofFSBA—treated solCD39 by the radio-TLC assay system demonstrated thatapprox. 94% of the enzymatic activity was blocked, while the phosphaterelease assay indicated that a similar percentage of the ATPase activitywas lost as well.

[0268] C. Mutagenesis Studies

[0269] To identify amino acids involved in the biological activity ofsolCD39, site directed mutagenesis was used to alter selected amino acidresidues in CD39. Mutants were assayed for enzymatic (ATPase and ADPase)and platelet inhibitory (dose-dependent inhibition of plateletaggregation) activities. For one series of mutants, residues within theconserved apyrase regions were replaced with alanine.

[0270] Platelet inhibitory activity correlated generally with enzymaticactivity. The E174A mutant (residues are numbered as in FIG. 1) wascompletely devoid of enzymatic activity and had no effect on plateletresponsiveness; the S218A mutant retained less than 10% of ADPaseactivity and approx. 10% of platelet inhibitory activity. Glutamate 174and Serine218 therefore appear to be important for both the enzymaticand platelet inhibitory activities of CD39.

[0271] Additional mutant forms of CD39 are expressed and assayed forenzymatic and platelet inhibitory activity in order to identify mutantswith increased or decreased activity as well as mutants thatpreferentially catalyze the ATPase or ADPase reaction.

Example 16

[0272] Persistence of solCD39 Following In Vivo Administration in Mice

[0273] Balb/c mice (6-8 weeks of age; maintained under specificpathogen-free conditions; Jackson Laboratory, Bar Harbor, Me.) wereintravenously injected with 50 μg recombinant solCD39 in 100 μl sterilesaline (0.9% NaCl). No overt external difficulties were noted in theanimals following injection. At various times after injection (5, 10, 30min, 1, 2, 4, 8, 24 h), pairs of mice were bled by cardiac puncture andeuthanized. Serum was prepared from each blood sample and frozen untilassay. The presence of biologically active solCD39 in serum samples wasmeasured in ATPase and ADPase assays. The data were fit using Deltagraph(Deltapoint, Monterey, Calif.). The best fit was derived using doubleexponential decay. Where indicated, specificity of enzyme activity wasdetermined by incubating serum samples with anti-CD39 mAb-coated beadsto remove CD39 prior to testing for ATPase activity.

[0274] As shown in FIG. 10, the data obtained best fit a biphasicexponential curve. The amount of ATPase activity from 25 μg/ml ofsolCD39 placed in murine serum is presented for comparison. The t_(½)α(distribution phase) was calculated to be 59 min in the ATPase assay and43 min in the ADPase assay. Approximately 55-65% of apyrase activity wascleared from the circulation during this phase. The elimination phasehad a t_(½)↑ of approximately 40 h in both assays. Preclearing the 10min, 2h, and 24 h time point samples with anti-CD39 mAb-coated beadscompletely eliminated serum ATPase/ADPase activities. These data alsodemonstrate that the assays specifically detect recombinant humansolCD39.

Example 17 Pilot Dose Ranging Study in Yorkshire-Hampshire Pigs

[0275] SolCD39 was administered to Yorkshire-Hampshire pigs, which havebeen developed as a porcine model of thrombosis. Following intravenousinjection, CD39 persisted in the circulation and was capable ofinhibiting platelet aggregation and recruitment for as much as a weekfollowing injection. This is in marked contrast to many othertherapeutic agents used for platelet inhibition, wherein the duration ofinhibition is very short.

[0276] Ten pigs were randomly assigned to receive solCD39 in low (72μg/kg), medium (221 μg/kg), or high (670 μg/kg) doses. Aspirin wasadministered orally on a daily basis. Placebo controls consisted ofaspirin. Saline controls and solCD39 were administered as a singlebolus. Time points were measured following this administration. Bloodsamples were obtained via an external jugular vein catheter. Bleedingtimes were measured in pigs receiving placebo controls and in thosereceiving solCD39 at baseline and at 60 minutes. ADP-induced plateletaggregation was measured at specific time intervals followingadministration. The concentration of CD39 in serum as a function of timewas measured using an ELISA assay.

[0277] Administration of solCD39 was well tolerated. It did not induceanemia or thrombocytopenia and, importantly, a second dose of solCD39could be administered without observable ill effects, such ashypotension, thrombocytopenia, or hemorrhage. Clot retraction was normalfollowing all experiments, indicating that platelet function wasessentially normal.

[0278] A. Effect of solCD39 on Bleeding Time

[0279] Bleeding time is an absolute measure of platelet function. Asshown in FIG. 11, solCD39 induced a prolonged bleeding time. Thisindicated that a therapeutic effect had been obtained via a mildinterference with platelet function. These mild increases in bleedingtime were similar to those obtained by aspirin administration. Thisindicates that the hemorrhagic defect was mild.

[0280] B. Effects of Aspirin and solCD39 on Platelet Aggregation

[0281]FIG. 12A shows the effect of aspirin on platelet aggregation atbaseline and at day 5, and FIG. 12B shows the effect of high dosesolCD39 on platelet aggregation at baseline and at day 7. Peak heightsfrom the platelet aggregation curves for each of the three solCD39 dosesare plotted in FIG. 13. The platelet aggregation data are also comparedby plotting relative areas from the platelet aggregation curves for eachof the three solCD39 doses. A dose of 670 μg/kg inhibited greater than90% of ADP induced platelet aggregation. The inhibitory effect waslong-lived, with 30% inhibition (after high dose solCD39) at two weeks.These experiments show that solCD39 has potent and long lastinganti-platelet effects, and that these effects are superior to thoseobtained using aspirin.

[0282] C. Persistence of solCD39 in Serum

[0283] The persistence of solCD39 in porcine serum, as determined byELISA, is shown in FIG. 14. Distribution and clearance half-lives weredetermined using a biphasic curve fit. The t_(½)α (distribution phase)was calculated to be 29 minutes. The elimination phase had a t_(½)β ofapproximately 51 hours. SolCD39 biological activity (ADPase activity)also exhibited a long elimination half-life, approaching 5-7 days, andcould still be detected over two weeks after administration. During thistime there were no changes in hematologic parameters and no evidence ofhemorrhage despite tripling of the bleeding time.

[0284] D. Percutaneous Transluminal Coronary Angioplasty (PTCA) Study

[0285] Porcine platelets and fibrinogen were labeled with ¹¹¹Indium and¹²⁵Iodine respectively for infusion into pigs. Twelve pigs were sedatedand anesthetized, and randomly assigned to receive intravenous solCD39(670 μg/kg) plus heparin and ASA or intravenous saline placebo plusheparin and ASA. Oral ASA was given to all pigs for at least three dayprior to the coronary angioplasty procedure, and heparin (100 U/kg) wasgiven at the time of the angioplasty. One to three days prior to theangioplasty an external jugular line was inserted to administer thelabeled platelets and fibrin, CD39 or saline, and to facilitate blooddraws. Labeled platelets and fibrinogen were given approximately 18hours prior to balloon injury. Coronary arteries were injured using anoversize balloon. A coronary guide catheter was first advanced into theascending aorta. An oversized balloon was then advanced into a coronaryvessel and inflated at 6 to 8 atmospheres for a total of thirty seconds.The balloon was then deflated and withdrawn. The average ratio ofballoon size to vessel size was 1.32 for the placebo group and 1.29 forthe CD39 group.

[0286] Platelet aggregation and bleeding time were measured 30 minutesafter administration. The pigs were killed 24 hours after balloon injuryand solCD39 administration, and the labeled platelet (¹¹¹Indium) andfibrin (¹²⁵Iodine) deposition per cm² was measured in the injuredcoronary artery segments. The results are summarized in TABLE 7. CD39administration was well tolerated without bleeding or hemodynamiccomplications. Moreover, no bleeding was noted during PTCA or aftersheath removal and there was no significant difference in hematocrit orplatelet counts between the groups.

[0287] These results show that the administration of solCD39 results ina significant inhibition of platelet aggregation and prolongation ofbleeding time, as well as a trend toward inhibition of platelet andfibrin deposition, after balloon injury in animals. The results alsosuggest that CD 39 has a minimal risk of inducing bleeding. TABLE 7Effects of solCD39 After Balloon Injury Platelet Fibrin % Inhibition ofDeposition Deposition Bleeding Platelet Treatment Ratio Ratio TimeAggregation Placebo 1.78 ± 0.4  0.71 ± 0.14 3.03 ± 0.2   1 ± 10 solCD391.25 ± 0.19 0.62 ± 0.10 7.00 ± 0.81 80 ± 2  p = value 0.2 0.5 0.0090.001

[0288] After the radioactivity decayed, toluidine-blue stained injuredcoronary artery segments were examined histologically, in order tofurther characterize the extent of thrombus formation. A blindedobserver qualitatively evaluated the degree of histologic injury in thecoronary segments by assessing, on a scale of 1-4 with 4 being the mostsevere injury, the severity of medial and internal elastic lamina tear,medial separation, and hemorrhage. A composite injury score was obtainedby totaling the three individual scores. The medial injury scores forthe placebo and CD39 groups were 2.5 and 2.2 respectively; medialseparation scores for the placebo and CD39 groups were 2.0 and 1.6respectively; the degree of hemorrhage for the placebo and CD39 groupswere 2.3 and 2.5 respectively. The composite injury scores for theplacebo and CD39 groups were 6.6 and 6.2 respectively. These in vivoresults correlate well with results, reported herein, obtained in vitroand ex vivo.

Example 18 Soluble CD39 Provides Additive Inhibition of PlateletAggregation Over Aspirin and Abciximab

[0289] An ex vivo study was performed in order to evaluate the additiveinhibition of platelet aggregation when soluble CD39 is added toplatelet rich plasma from patients receiving: placebo, aspirin,clopidogrel, ticlopidine, or abciximab. Each group consisted of three tosix patients. The clopidogrel, ticlopidine, and abciximab groups alsoreceived aspirin. Baseline platelet aggregation was measured for eachgroup, in response to the platelet agonists ADP, collagen, or theThrombin Receptor Activating Peptide TRAP₁₋₆. SoICD39 (10 μg/ml or 100μg/ml) was then added and the additional inhibition of plateletactivation (over baseline, in response to the platelet agonists) wasmeasured in each of the five groups. The result are shown in TABLE 8.

[0290] Soluble CD39 at a concentration of 10 μg/ml synergisticallyinhibited ADP, collagen, and TRAP mediated platelet aggregation inpatients on aspirin (p<0.001), and this effect was independent ofclopidogrel and ticlopidine. Abciximab alone abolished plateletaggregation due to ADP and collagen, but CD39 provided synergisticinhibition of platelet aggregation induced by TRAP (p<0.007). SolubleCD39 at 100 μg/ml provided increased inhibition of platelet aggregationto all agonists. These results were also seen in vitro. Collagen andTRAP induce platelet aggregation via mechanisms in addition to ADPrelease and recruitment, so the ability of CD39 to inhibit collagen andTRAP-mediated platelet aggregation suggests additional versatility ofCD39 as an antithrombotic agent. TABLE 8 Additive Inhibition of PlateletAggregation by Soluble CD39 Group Placebo Aspirin ClopidogrelTiclopidine Abciximab Baseline ADP 84 ± 4¹ 69 ± 5 58 ± 6 76 ± 3  0 ± 0Collagen 85 ± 1 62 ± 8 57 ± 9 71 ± 17  0 ± 0 TRAP 94 ± 2 66 ± 6 51 ± 226 ± 5 46 ± 6 SolCD39  10 μg/ml ADP  0 ± 0  4 ± 2  5 ± 2 10 ± 2  0 ± 0100%² 97%  92% 86% 100% Collagen 75 ± 2 21 ± 4 31 ± 9 48 ± 18  0 ± 0 11% 68%  48% 37% 100% TRAP 70 ± 3 38 ± 7 35 ± 1 19 ± 6 22 ± 10  26% 45% 31% 33%  52% SolCD39 100 μg/ml ADP  0 ± 0  1 ± 0  0 ± 0  2 ± 2  0 ± 0100% 99% 100% 97% 100% Collagen 57 ± 5 16 ± 4 21 ± 6 37 ± 15  0 ± 0  33%75%  64% 53% 100% TRAP 65 ± 4 26 ± 5 23 ± 3 18 ± 7 22 ± 9  30% 63%  55%36%  52%

Example 19

[0291] Soluble CD39 Inhibits Thrombosis and Limits Ischemic CerebralInjury in Wild Type and Reconstituted CD39 Null Mice

[0292] The above examples suggested that soluble CD39 would inhibitADP-mediated amplification of platelet recruitment in distalmicrovessels, thereby reducing thrombosis after stroke. The followingexperiments illustrate the use of CD39 in a microvascular thrombosis(murine ischemic stroke) model. Soluble CD39 inhibited microvascularthrombosis and conferred cerebroprotection in stroke. A notable featureof the solCD39 treatment was the low incidence of intracerebralhemorrhage relative to that reported for other antithrombotic agents.

[0293] A. Materials and Methods

[0294] C57BL/6J mice (6-8 wk) were obtained from Jackson Laboratories(Bar Harbor, Me.). Untreated mice, and mice treated with 4 mg/kgsolCD39, with 5 mg/kg aspirin or phosphate buffered saline, wereanesthetized and heparinized (10 U/g), prior to blood collection viacardiac puncture. 80 μL of 3.8% trisodium citrate was added to each mLof blood. Samples from 6-8 mice were pooled and platelet-rich plasma(PRP) was prepared by centrifugation. The PRP contained 400-700×10³platelets per μL. All experiments were completed within 2 hours of bloodcollection. PRP (200 μL) was preincubated, for 3 min. at 37° C., with100 μL Tris-buffered saline buffer (15 mM NaCl, 5 mM glucose, pH 7.4) inan aggregometer cuvette (Lumiaggregometer; Chrono-Log, Havertown, Pa.),and the platelet agonists ADP, collagen, or sodium arachidonate wereadded at the final concentrations indicated. Aggregation responses wererecorded for 2-4 min, and expressed as area under the curve (heighttimes width at ½ height).

[0295] The effects of soluble CD39 were tested in a previously validatedmurine model of stroke injury (Choudhri, T. F., et al., J. Clin. Invest.102:1301-1310 (1998); Connolly, E. S., Jr., et al., J. Clin. Invest.97:209-216 (1996); and Connolly, E. S., Jr., et al., Neurosurg.38(3):523-532 (1996)). Anesthetized mice were maintained at 37±2° C.during and for 90 min following surgery. A midline neck incision wasmade and the right carotid artery exposed. Middle cerebral arteryocclusion was accomplished by advancing a 13-mm heat-blunt tipped 6-0nylon suture via an arteriotomy in the external carotid stump. Theexternal carotid artery was cauterized to secure hemostasis, andarterial flow re-established. Carotid artery occlusion never exceed 3min. The occluding suture was removed after 45 min and cautery was againlocally applied to prevent bleeding at the arteriotomy site. Surgicalstaples were used for wound closure.

[0296] Doppler measurement of cerebral cortical blood flow, neurologicalscore (Huang, Z., et al., Science 265:183-1885 (1994)), calculation ofinfarct volume, measurement of cerebral thrombosis using ¹¹¹In-labeledplatelets (Choudhri, T. F., et al., J. Clin. Invest. 102:1301-1310(1998) and Naka, Y., et al., Circ. Res. 76:900-906 (1995)), detection ofintracerebral fibrin (Choudhri, T. F., et al., J. Clin. Invest.102:1301-1310 (1998)), and measurement of intracerebral hemorrhage(Choudhri, T. F., et al., J. Clin. Invest. 102:1301-1310 (1998) andChoudhri, T.F., et al., Stroke 28:2296-2302 (1997)) were measured aspreviously described. The results are described below.

[0297] B. Soluble CD39 Abrogates the Ex Vivo Aggregation of MurinePlatelets

[0298] Platelet-rich plasma was obtained from mice 1 hour afterinjection of saline (vehicle), soluble CD39, or aspirin. Ex vivoplatelet aggregation was studied to ascertain the relative potency ofsolCD39 as compared to aspirin (which can improve the outcome followinga transient ischemic attack). Platelets from control and aspirin-treatedmice strongly aggregated following stimulation with ADP (FIG. 15A) orcollagen (FIG. 15B).

[0299] Soluble CD39 abrogated platelet aggregation in the presence ofADP, and attenuated aggregation in the presence of collagen andarachidonate. In contrast, aspirin treatment only blocked plateletreactivity to arachidonate (FIG. 15C). The platelets from micepretreated with solCD39 showed an initial aggregation in the presence ofarachidonate, but rapidly disaggregated and returned to the restingstate before a full response occurred (FIG. 15C).

[0300] C. Soluble CD39 Is Effective Even When Added at the Peak of theAggregation Response

[0301] ADP (5 μM) was added to mouse platelets in vitro to induce anaggregation response. Soluble CD39 (2.5 μg/ml or 1.25 μg/ml) was addedat the peak of the aggregation response. The solCD39 immediatelyreversed the aggregation response, as shown in FIG. 16. This resultdemonstrates that SolCD39 is able to reverse an aggregation response,rapidly returning platelets to a resting state, even when added at thepeak of the response. This result likely reflects the fact that at thepeak of the aggregation response ADP is prominent in the releasate fromthe aggregating platelets. Soluble CD39 metabolizes this ADP to thebiologically inactive compound AMP almost instantaneously, accountingfor the rapid descent of the aggregation curve in FIG. 16, right side.

[0302] D. Soluble CD39 Reduces the Sequelae of Stroke

[0303] Intravenously injected soluble CD39 showed therapeutic utility instroke. Soluble CD39 inhibited platelet accumulation in the ipsilateralcerebral hemisphere following induction of stroke, as shown in FIG. 17A.Similarly, solCD39 decreased the level of fibrin accumulation in theipsilateral hemisphere (vs. contralateral) as measured by Western blotanalysis using a fibrin specific antibody (FIG. 17B).

[0304] The ability of solCD39 to reduce thrombosis, as measured bydecreased platelet and fibrin deposition, was accompanied by improvedpostischemic cerebral perfusion 24 hours after stroke induction, asshown in FIG. 18A. In contrast, when aspirin was administered at aclinically relevant dose (that inhibited the ex vivo response ofplatelets to arachidonate) no improvement was seen in postischemiccerebral blood flow (FIG. 18A).

[0305] Preoperatively administered solCD39 conferred a dose-dependentdiminution of cerebral infarct volume, as measured by digitalhistological analysis (FIG. 18B). Aspirin, in contrast, showed atendency to decrease cerebral infarct volume, although this effect wasnot statistically significant. The administration of soluble CD39 eitherprior to, or up to 3 h following, stroke reduced both neurologicaldeficit (FIG. 18C) and mortality (FIG. 18D).

[0306] The effects of soluble CD39 and aspirin on the development ofintracerebral hemorrhage following stroke are shown in FIG. 18E. Aspirinincreased intracerebral hemorrhage (as measured spectrophotometrically)significantly, but there was no significant increase in intracerebralhemorrhage at any dose of soluble CD39 tested. At these doses, solubleCD39 inhibited both platelet and fibrin accumulation and promoted anincrease in postischemic blood flow, as shown in FIGS. 17A, 17B, and18A. FIG. 19 shows a covariate plot of cerebral infarct volume vs.intracerebral hemorrhage for each treatment, and indicates that aspirinis less capable of reducing infarct volume and preventing intracerebralhemorrhage than soluble CD39. In summary, at the doses tested in themouse stroke model, solCD39 conferred protection without inducing thebleeding problems that often accompany anti-thrombotic therapy regimens.

[0307] E. CD39 Null Mice Can be Reconstituted with Soluble CD39

[0308] CD39-/- mice were generated using a gene targeting vector inwhich exons 4-6, encoding apyrase conserved regions 2-4 (Handa, M. &Guidotti, G., Biochem. Biophys. Res. Commun. 218:916-923 (1996); Wang,T. F. & Guidotti, G., J. BioL Chem. 271:9898-9901 (1996); Maliszewski,C. R., et al., J. Immunol. 153:3574-3583 (1994); and Schoenborn, M. A.,et al., Cytogen Cell Gen. 81(3-4):287-280 (1998)), were replaced with aPGKneo cassette, as shown in FIG. 20A. The gene targeting vector, inwhich a 4.1 kb SpeI-BglII fragment containing exons 4-6 was replacedwith a PGKneo cassette, was introduced into 129-derived ES cells. Cellswere selected in G418 and gancyclovir. Nine ES clones with a disruptedCD39 allele, as identified by genomic Southern blot analyses of BglIIdigested DNA as shown in FIG. 20B, were injected into blastocysts andthe resulting chimeras crossed to C57BL/6 to produceCD39+/−heterozygotes. CD39-/- mice were generated at the expectedMendelian frequency from CD39+/−intercrosses. The CD39-/- mice used inthe experiments described below represent random C57BL/6×129 hybrids.

[0309] Homozygous CD39-/- mice were overtly normal, and did not displayan obvious phenotype in the unperturbed state. Hematological profiles,including erythrocyte parameters, platelet counts, leukocyte counts anddifferentials, and coagulation screening, were normal. As shown below,the CD39-null mice did not exhibit a prothrombotic phenotype unlesschallenged by experimental stroke. Under those conditions, the defectwas abolished and normal blood fluidity was restored by administrationof soluble CD39. Bleeding times of CD39-/- mice were normal, indicatingthat normal blood flow in an unperturbed animal is not dependent uponendogenous expression of CD39. As is seen in normal mice, CD394- animalsexhibited markedly increased bleeding times following the administrationof aspirin or following administration of increasing doses of solCD39 asshown in FIG. 21. CD39-/- mice subjected to focal cerebral ischemiaexhibited diminished blood flow following reperfusion as compared togenetically matched controls (FIG. 22A), indicating that endogenous CD39contributes to maintenance of hemostasis during episodes of vascularinjury. When solCD39 (8 mg/kg) was administered to the CD39-/- mice,these mice were “reconstituted” as shown by a postischemic blood flowsimilar to untreated controls. CD39-/- mice demonstrated increasedcerebral infarction volume as compared to genotype-matched controlsfollowing induced stroke (FIG. 22B). CD39-/- mice “reconstituted” withsolCD39 had markedly diminished infarct volume, indicating a protectiveeffect of solCD39. Other parameters (neurological deficit scores,overall mortality, and intracerebral hemorrhage) did not differ betweengroups (FIG. 22 C, D, E).

[0310] These results demonstrate that CD39 inhibits microvascularthrombosis and confers cerebroprotection without inducing intracerebralhemorrhage in a murine model of stroke. Soluble CD39 decreased plateletdeposition, fibrin deposition, and cerebral infarction volume. SolubleCD39 reduced infarction volume and restored postischemic blood flow evenwhen administered three hours following stroke induction. This result isimportant because the average patient experiencing a stroke appears inthe emergency room approximately three hours after the initial eventoccurs. The ability to treat patients with solCD39 after three hoursprovides an important advantage over many other agents designed toinhibit platelet reactivity.

Example 20 Soluble CD39 Improves Survival in a Mouse Ischemia Model

[0311] C57BL/6 mice were anesthetized and ventilated, and their thoraceswere opened to surgically expose both pulmonary hila. Eitherphysiological saline or soluble CD39 (8 mg/kg) was administeredintravenously, after which the left pulmonary hilum was cross-clampedfor one hour. The cross-clamp was removed for three hours ofreperfusion, and then a cross-clamp was applied to the right hilum for athirty minute observation period. This latter maneuver effectivelyremoved the normal lung from circulation, so that the mouse must surviveon the function of the post-ischemic left lung. The results are shown,in the form of a Kaplan-Meier survival plot, in FIG. 23. All of the micegiven saline (n=6) died prior to the thirty minute time point whereasall of the sol39-treated mice (n=3) survived for thirty minutes.

[0312] The long lasting effects of soluble CD39 are also shown to beclinically useful in the reduction of complications of atherosclerosis,such as myocardial infarction, stroke, and peripheral vascularocclusion. Patients suffering from these conditions demonstrate anabundance of activated platelets in their circulation, and suchactivated platelets have a lowered threshold for ADP stimulation.Soluble CD39 metabolically deletes ADP from the fluid phase of activatedplatelets and reverses their prothrombotic characteristics.

[0313] The relevant disclosures of publications cited herein arespecifically incorporated by reference. The examples presented above arenot intended to be exhaustive or to limit the scope of the invention.The skilled artisan will understand that variations and modificationsand variations are possible in light of the above teachings, and suchmodifications and variations are intended to be within the scope of theinvention.

1 31 1 1599 DNA Homo sapiens CDS (67)..(1596) 1 ccacaccaag cagcggctgggggggggaaa gacgaggaaa gaggaggaaa acaaaagctg 60 ctactt atg gaa gat acaaag gag tct aac gtg aag aca ttt tgc tcc 108 Met Glu Asp Thr Lys Glu SerAsn Val Lys Thr Phe Cys Ser 1 5 10 aag aat atc cta gcc atc ctt ggc ttctcc tct atc ata gct gtg ata 156 Lys Asn Ile Leu Ala Ile Leu Gly Phe SerSer Ile Ile Ala Val Ile 15 20 25 30 gct ttg ctt gct gtg ggg ttg acc cagaac aaa gca ttg cca gaa aac 204 Ala Leu Leu Ala Val Gly Leu Thr Gln AsnLys Ala Leu Pro Glu Asn 35 40 45 gtt aag tat ggg att gtg ctg gat gcg ggttct tct cac aca agt tta 252 Val Lys Tyr Gly Ile Val Leu Asp Ala Gly SerSer His Thr Ser Leu 50 55 60 tac atc tat aag tgg cca gca gaa aag gag aatgac aca ggc gtg gtg 300 Tyr Ile Tyr Lys Trp Pro Ala Glu Lys Glu Asn AspThr Gly Val Val 65 70 75 cat caa gta gaa gaa tgc agg gtt aaa ggt cct ggaatc tca aaa ttt 348 His Gln Val Glu Glu Cys Arg Val Lys Gly Pro Gly IleSer Lys Phe 80 85 90 gtt cag aaa gta aat gaa ata ggc att tac ctg act gattgc atg gaa 396 Val Gln Lys Val Asn Glu Ile Gly Ile Tyr Leu Thr Asp CysMet Glu 95 100 105 110 aga gct agg gaa gtg att cca agg tcc cag cac caagag aca ccc gtt 444 Arg Ala Arg Glu Val Ile Pro Arg Ser Gln His Gln GluThr Pro Val 115 120 125 tac ctg gga gcc acg gca ggc atg cgg ttg ctc aggatg gaa agt gaa 492 Tyr Leu Gly Ala Thr Ala Gly Met Arg Leu Leu Arg MetGlu Ser Glu 130 135 140 gag ttg gca gac agg gtt ctg gat gtg gtg gag aggagc ctc agc aac 540 Glu Leu Ala Asp Arg Val Leu Asp Val Val Glu Arg SerLeu Ser Asn 145 150 155 tac ccc ttt gac ttc cag ggt gcc agg atc att actggc caa gag gaa 588 Tyr Pro Phe Asp Phe Gln Gly Ala Arg Ile Ile Thr GlyGln Glu Glu 160 165 170 ggt gcc tat ggc tgg att act atc aac tat ctg ctgggc aaa ttc agt 636 Gly Ala Tyr Gly Trp Ile Thr Ile Asn Tyr Leu Leu GlyLys Phe Ser 175 180 185 190 cag aaa aca agg tgg ttc agc ata gtc cca tatgaa acc aat aat cag 684 Gln Lys Thr Arg Trp Phe Ser Ile Val Pro Tyr GluThr Asn Asn Gln 195 200 205 gaa acc ttt gga gct ttg gac ctt ggg gga gcctct aca caa gtc act 732 Glu Thr Phe Gly Ala Leu Asp Leu Gly Gly Ala SerThr Gln Val Thr 210 215 220 ttt gta ccc caa aac cag act atc gag tcc ccagat aat gct ctg caa 780 Phe Val Pro Gln Asn Gln Thr Ile Glu Ser Pro AspAsn Ala Leu Gln 225 230 235 ttt cgc ctc tat ggc aag gac tac aat gtc tacaca cat agc ttc ttg 828 Phe Arg Leu Tyr Gly Lys Asp Tyr Asn Val Tyr ThrHis Ser Phe Leu 240 245 250 tgc tat ggg aag gat cag gca ctc tgg cag aaactg gcc aag gac att 876 Cys Tyr Gly Lys Asp Gln Ala Leu Trp Gln Lys LeuAla Lys Asp Ile 255 260 265 270 cag gtt gca agt aat gaa att ctc agg gaccca tgc ttt cat cct gga 924 Gln Val Ala Ser Asn Glu Ile Leu Arg Asp ProCys Phe His Pro Gly 275 280 285 tat aag aag gta gtg aac gta agt gac ctttac aag acc ccc tgc acc 972 Tyr Lys Lys Val Val Asn Val Ser Asp Leu TyrLys Thr Pro Cys Thr 290 295 300 aag aga ttt gag atg act ctt cca ttc cagcag ttt gaa atc cag ggt 1020 Lys Arg Phe Glu Met Thr Leu Pro Phe Gln GlnPhe Glu Ile Gln Gly 305 310 315 att gga aac tat caa caa tgc cat caa agcatc ctg gag ctc ttc aac 1068 Ile Gly Asn Tyr Gln Gln Cys His Gln Ser IleLeu Glu Leu Phe Asn 320 325 330 acc agt tac tgc cct tac tcc cag tgt gccttc aat ggg att ttc ttg 1116 Thr Ser Tyr Cys Pro Tyr Ser Gln Cys Ala PheAsn Gly Ile Phe Leu 335 340 345 350 cca cca ctc cag ggg gat ttt ggg gcattt tca gct ttt tac ttt gtg 1164 Pro Pro Leu Gln Gly Asp Phe Gly Ala PheSer Ala Phe Tyr Phe Val 355 360 365 atg aag ttt tta aac ttg aca tca gagaaa gtc tct cag gaa aag gtg 1212 Met Lys Phe Leu Asn Leu Thr Ser Glu LysVal Ser Gln Glu Lys Val 370 375 380 act gag atg atg aaa aag ttc tgt gctcag cct tgg gag gag ata aaa 1260 Thr Glu Met Met Lys Lys Phe Cys Ala GlnPro Trp Glu Glu Ile Lys 385 390 395 aca tct tac gct gga gta aag gag aagtac ctg agt gaa tac tgc ttt 1308 Thr Ser Tyr Ala Gly Val Lys Glu Lys TyrLeu Ser Glu Tyr Cys Phe 400 405 410 tct ggt acc tac att ctc tcc ctc cttctg caa ggc tat cat ttc aca 1356 Ser Gly Thr Tyr Ile Leu Ser Leu Leu LeuGln Gly Tyr His Phe Thr 415 420 425 430 gct gat tcc tgg gag cac atc catttc att ggc aag atc cag ggc agc 1404 Ala Asp Ser Trp Glu His Ile His PheIle Gly Lys Ile Gln Gly Ser 435 440 445 gac gcc ggc tgg act ttg ggc tacatg ctg aac ctg acc aac atg atc 1452 Asp Ala Gly Trp Thr Leu Gly Tyr MetLeu Asn Leu Thr Asn Met Ile 450 455 460 cca gct gag caa cca ttg tcc acacct ctc tcc cac tcc acc tat gtc 1500 Pro Ala Glu Gln Pro Leu Ser Thr ProLeu Ser His Ser Thr Tyr Val 465 470 475 ttc ctc atg gtt cta ttc tcc ctggtc ctt ttc aca gtg gcc atc ata 1548 Phe Leu Met Val Leu Phe Ser Leu ValLeu Phe Thr Val Ala Ile Ile 480 485 490 ggc ttg ctt atc ttt cac aag ccttca tat ttc tgg aaa gat atg gta 1596 Gly Leu Leu Ile Phe His Lys Pro SerTyr Phe Trp Lys Asp Met Val 495 500 505 510 tag 1599 2 510 PRT Homosapiens 2 Met Glu Asp Thr Lys Glu Ser Asn Val Lys Thr Phe Cys Ser LysAsn 1 5 10 15 Ile Leu Ala Ile Leu Gly Phe Ser Ser Ile Ile Ala Val IleAla Leu 20 25 30 Leu Ala Val Gly Leu Thr Gln Asn Lys Ala Leu Pro Glu AsnVal Lys 35 40 45 Tyr Gly Ile Val Leu Asp Ala Gly Ser Ser His Thr Ser LeuTyr Ile 50 55 60 Tyr Lys Trp Pro Ala Glu Lys Glu Asn Asp Thr Gly Val ValHis Gln 65 70 75 80 Val Glu Glu Cys Arg Val Lys Gly Pro Gly Ile Ser LysPhe Val Gln 85 90 95 Lys Val Asn Glu Ile Gly Ile Tyr Leu Thr Asp Cys MetGlu Arg Ala 100 105 110 Arg Glu Val Ile Pro Arg Ser Gln His Gln Glu ThrPro Val Tyr Leu 115 120 125 Gly Ala Thr Ala Gly Met Arg Leu Leu Arg MetGlu Ser Glu Glu Leu 130 135 140 Ala Asp Arg Val Leu Asp Val Val Glu ArgSer Leu Ser Asn Tyr Pro 145 150 155 160 Phe Asp Phe Gln Gly Ala Arg IleIle Thr Gly Gln Glu Glu Gly Ala 165 170 175 Tyr Gly Trp Ile Thr Ile AsnTyr Leu Leu Gly Lys Phe Ser Gln Lys 180 185 190 Thr Arg Trp Phe Ser IleVal Pro Tyr Glu Thr Asn Asn Gln Glu Thr 195 200 205 Phe Gly Ala Leu AspLeu Gly Gly Ala Ser Thr Gln Val Thr Phe Val 210 215 220 Pro Gln Asn GlnThr Ile Glu Ser Pro Asp Asn Ala Leu Gln Phe Arg 225 230 235 240 Leu TyrGly Lys Asp Tyr Asn Val Tyr Thr His Ser Phe Leu Cys Tyr 245 250 255 GlyLys Asp Gln Ala Leu Trp Gln Lys Leu Ala Lys Asp Ile Gln Val 260 265 270Ala Ser Asn Glu Ile Leu Arg Asp Pro Cys Phe His Pro Gly Tyr Lys 275 280285 Lys Val Val Asn Val Ser Asp Leu Tyr Lys Thr Pro Cys Thr Lys Arg 290295 300 Phe Glu Met Thr Leu Pro Phe Gln Gln Phe Glu Ile Gln Gly Ile Gly305 310 315 320 Asn Tyr Gln Gln Cys His Gln Ser Ile Leu Glu Leu Phe AsnThr Ser 325 330 335 Tyr Cys Pro Tyr Ser Gln Cys Ala Phe Asn Gly Ile PheLeu Pro Pro 340 345 350 Leu Gln Gly Asp Phe Gly Ala Phe Ser Ala Phe TyrPhe Val Met Lys 355 360 365 Phe Leu Asn Leu Thr Ser Glu Lys Val Ser GlnGlu Lys Val Thr Glu 370 375 380 Met Met Lys Lys Phe Cys Ala Gln Pro TrpGlu Glu Ile Lys Thr Ser 385 390 395 400 Tyr Ala Gly Val Lys Glu Lys TyrLeu Ser Glu Tyr Cys Phe Ser Gly 405 410 415 Thr Tyr Ile Leu Ser Leu LeuLeu Gln Gly Tyr His Phe Thr Ala Asp 420 425 430 Ser Trp Glu His Ile HisPhe Ile Gly Lys Ile Gln Gly Ser Asp Ala 435 440 445 Gly Trp Thr Leu GlyTyr Met Leu Asn Leu Thr Asn Met Ile Pro Ala 450 455 460 Glu Gln Pro LeuSer Thr Pro Leu Ser His Ser Thr Tyr Val Phe Leu 465 470 475 480 Met ValLeu Phe Ser Leu Val Leu Phe Thr Val Ala Ile Ile Gly Leu 485 490 495 LeuIle Phe His Lys Pro Ser Tyr Phe Trp Lys Asp Met Val 500 505 510 3 476PRT Artificial Sequence Description of Artificial Sequence Fusionconstruct of human CD39 3 Met Ala Thr Ser Trp Gly Thr Val Phe Phe MetLeu Val Val Ser Cys 1 5 10 15 Val Cys Ser Ala Val Ser His Arg Asn GlnGln Thr Trp Phe Glu Gly 20 25 30 Ile Phe Leu Ser Ser Thr Gln Asn Lys AlaLeu Pro Glu Asn Val Lys 35 40 45 Tyr Gly Ile Val Leu Asp Ala Gly Ser SerHis Thr Ser Leu Tyr Ile 50 55 60 Tyr Lys Trp Pro Ala Glu Lys Glu Asn AspThr Gly Val Val His Gln 65 70 75 80 Val Glu Glu Cys Arg Val Lys Gly ProGly Ile Ser Lys Phe Val Gln 85 90 95 Lys Val Asn Glu Ile Gly Ile Tyr LeuThr Asp Cys Met Glu Arg Ala 100 105 110 Arg Glu Val Ile Pro Arg Ser GlnHis Gln Glu Thr Pro Val Tyr Leu 115 120 125 Gly Ala Thr Ala Gly Met ArgLeu Leu Arg Met Glu Ser Glu Glu Leu 130 135 140 Ala Asp Arg Val Leu AspVal Val Glu Arg Ser Leu Ser Asn Tyr Pro 145 150 155 160 Phe Asp Phe GlnGly Ala Arg Ile Ile Thr Gly Gln Glu Glu Gly Ala 165 170 175 Tyr Gly TrpIle Thr Ile Asn Tyr Leu Leu Gly Lys Phe Ser Gln Lys 180 185 190 Thr ArgTrp Phe Ser Ile Val Pro Tyr Glu Thr Asn Asn Gln Glu Thr 195 200 205 PheGly Ala Leu Asp Leu Gly Gly Ala Ser Thr Gln Val Thr Phe Val 210 215 220Pro Gln Asn Gln Thr Ile Glu Ser Pro Asp Asn Ala Leu Gln Phe Arg 225 230235 240 Leu Tyr Gly Lys Asp Tyr Asn Val Tyr Thr His Ser Phe Leu Cys Tyr245 250 255 Gly Lys Asp Gln Ala Leu Trp Gln Lys Leu Ala Lys Asp Ile GlnVal 260 265 270 Ala Ser Asn Glu Ile Leu Arg Asp Pro Cys Phe His Pro GlyTyr Lys 275 280 285 Lys Val Val Asn Val Ser Asp Leu Tyr Lys Thr Pro CysThr Lys Arg 290 295 300 Phe Glu Met Thr Leu Pro Phe Gln Gln Phe Glu IleGln Gly Ile Gly 305 310 315 320 Asn Tyr Gln Gln Cys His Gln Ser Ile LeuGlu Leu Phe Asn Thr Ser 325 330 335 Tyr Cys Pro Tyr Ser Gln Cys Ala PheAsn Gly Ile Phe Leu Pro Pro 340 345 350 Leu Gln Gly Asp Phe Gly Ala PheSer Ala Phe Tyr Phe Val Met Lys 355 360 365 Phe Leu Asn Leu Thr Ser GluLys Val Ser Gln Glu Lys Val Thr Glu 370 375 380 Met Met Lys Lys Phe CysAla Gln Pro Trp Glu Glu Ile Lys Thr Ser 385 390 395 400 Tyr Ala Gly ValLys Glu Lys Tyr Leu Ser Glu Tyr Cys Phe Ser Gly 405 410 415 Thr Tyr IleLeu Ser Leu Leu Leu Gln Gly Tyr His Phe Thr Ala Asp 420 425 430 Ser TrpGlu His Ile His Phe Ile Gly Lys Ile Gln Gly Ser Asp Ala 435 440 445 GlyTrp Thr Leu Gly Tyr Met Leu Asn Leu Thr Asn Met Ile Pro Ala 450 455 460Glu Gln Pro Leu Ser Thr Pro Leu Ser His Ser Thr 465 470 475 4 476 PRTArtificial Sequence Description of Artificial Sequence Fusion constructof human CD39 4 Met Ala Thr Ser Trp Gly Thr Val Phe Phe Met Leu Val ValSer Cys 1 5 10 15 Val Cys Ser Ala Val Ser His Arg Asn Gln Gln Thr TrpPhe Glu Gly 20 25 30 Ile Phe Leu Ser Ser Met Xaa Pro Ile Asn Val Ser AlaSer Thr Leu 35 40 45 Tyr Gly Ile Val Leu Asp Ala Gly Ser Ser His Thr SerLeu Tyr Ile 50 55 60 Tyr Lys Trp Pro Ala Glu Lys Glu Asn Asp Thr Gly ValVal His Gln 65 70 75 80 Val Glu Glu Cys Arg Val Lys Gly Pro Gly Ile SerLys Phe Val Gln 85 90 95 Lys Val Asn Glu Ile Gly Ile Tyr Leu Thr Asp CysMet Glu Arg Ala 100 105 110 Arg Glu Val Ile Pro Arg Ser Gln His Gln GluThr Pro Val Tyr Leu 115 120 125 Gly Ala Thr Ala Gly Met Arg Leu Leu ArgMet Glu Ser Glu Glu Leu 130 135 140 Ala Asp Arg Val Leu Asp Val Val GluArg Ser Leu Ser Asn Tyr Pro 145 150 155 160 Phe Asp Phe Gln Gly Ala ArgIle Ile Thr Gly Gln Glu Glu Gly Ala 165 170 175 Tyr Gly Trp Ile Thr IleAsn Tyr Leu Leu Gly Lys Phe Ser Gln Lys 180 185 190 Thr Arg Trp Phe SerIle Val Pro Tyr Glu Thr Asn Asn Gln Glu Thr 195 200 205 Phe Gly Ala LeuAsp Leu Gly Gly Ala Ser Thr Gln Val Thr Phe Val 210 215 220 Pro Gln AsnGln Thr Ile Glu Ser Pro Asp Asn Ala Leu Gln Phe Arg 225 230 235 240 LeuTyr Gly Lys Asp Tyr Asn Val Tyr Thr His Ser Phe Leu Cys Tyr 245 250 255Gly Lys Asp Gln Ala Leu Trp Gln Lys Leu Ala Lys Asp Ile Gln Val 260 265270 Ala Ser Asn Glu Ile Leu Arg Asp Pro Cys Phe His Pro Gly Tyr Lys 275280 285 Lys Val Val Asn Val Ser Asp Leu Tyr Lys Thr Pro Cys Thr Lys Arg290 295 300 Phe Glu Met Thr Leu Pro Phe Gln Gln Phe Glu Ile Gln Gly IleGly 305 310 315 320 Asn Tyr Gln Gln Cys His Gln Ser Ile Leu Glu Leu PheAsn Thr Ser 325 330 335 Tyr Cys Pro Tyr Ser Gln Cys Ala Phe Asn Gly IlePhe Leu Pro Pro 340 345 350 Leu Gln Gly Asp Phe Gly Ala Phe Ser Ala PheTyr Phe Val Met Lys 355 360 365 Phe Leu Asn Leu Thr Ser Glu Lys Val SerGln Glu Lys Val Thr Glu 370 375 380 Met Met Lys Lys Phe Cys Ala Gln ProTrp Glu Glu Ile Lys Thr Ser 385 390 395 400 Tyr Ala Gly Val Lys Glu LysTyr Leu Ser Glu Tyr Cys Phe Ser Gly 405 410 415 Thr Tyr Ile Leu Ser LeuLeu Leu Gln Gly Tyr His Phe Thr Ala Asp 420 425 430 Ser Trp Glu His IleHis Phe Ile Gly Lys Ile Gln Gly Ser Asp Ala 435 440 445 Gly Trp Thr LeuGly Tyr Met Leu Asn Leu Thr Asn Met Ile Pro Ala 450 455 460 Glu Gln ProLeu Ser Thr Pro Leu Ser His Ser Thr 465 470 475 5 1365 DNA ArtificialSequence Description of Artificial Sequence Fusion construct of humanCD39 5 gca cct act tca agt tct aca aag aaa aca cag cta act agt tca acc48 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Thr Ser Ser Thr 1 510 15 cag aac aaa gca ttg cca gaa aac gtt aag tat ggg att gtg ctg gat 96Gln Asn Lys Ala Leu Pro Glu Asn Val Lys Tyr Gly Ile Val Leu Asp 20 25 30gcg ggt tct tct cac aca agt tta tac atc tat aag tgg cca gca gaa 144 AlaGly Ser Ser His Thr Ser Leu Tyr Ile Tyr Lys Trp Pro Ala Glu 35 40 45 aaggag aat gac aca ggc gtg gtg cat caa gta gaa gaa tgc agg gtt 192 Lys GluAsn Asp Thr Gly Val Val His Gln Val Glu Glu Cys Arg Val 50 55 60 aaa ggtcct gga atc tca aaa ttt gtt cag aaa gta aat gaa ata ggc 240 Lys Gly ProGly Ile Ser Lys Phe Val Gln Lys Val Asn Glu Ile Gly 65 70 75 80 att tacctg act gat tgc atg gaa aga gct agg gaa gtg att cca agg 288 Ile Tyr LeuThr Asp Cys Met Glu Arg Ala Arg Glu Val Ile Pro Arg 85 90 95 tcc cag caccaa gag aca ccc gtt tac ctg gga gcc acg gca ggc atg 336 Ser Gln His GlnGlu Thr Pro Val Tyr Leu Gly Ala Thr Ala Gly Met 100 105 110 cgg ttg ctcagg atg gaa agt gaa gag ttg gca gac agg gtt ctg gat 384 Arg Leu Leu ArgMet Glu Ser Glu Glu Leu Ala Asp Arg Val Leu Asp 115 120 125 gtg gtg gagagg agc ctc agc aac tac ccc ttt gac ttc cag ggt gcc 432 Val Val Glu ArgSer Leu Ser Asn Tyr Pro Phe Asp Phe Gln Gly Ala 130 135 140 agg atc attact ggc caa gag gaa ggt gcc tat ggc tgg att act atc 480 Arg Ile Ile ThrGly Gln Glu Glu Gly Ala Tyr Gly Trp Ile Thr Ile 145 150 155 160 aac tatctg ctg ggc aaa ttc agt cag aaa aca agg tgg ttc agc ata 528 Asn Tyr LeuLeu Gly Lys Phe Ser Gln Lys Thr Arg Trp Phe Ser Ile 165 170 175 gtc ccatat gaa acc aat aat cag gaa acc ttt gga gct ttg gac ctt 576 Val Pro TyrGlu Thr Asn Asn Gln Glu Thr Phe Gly Ala Leu Asp Leu 180 185 190 ggg ggagcc tct aca caa gtc act ttt gta ccc caa aac cag act atc 624 Gly Gly AlaSer Thr Gln Val Thr Phe Val Pro Gln Asn Gln Thr Ile 195 200 205 gag tcccca gat aat gct ctg caa ttt cgc ctc tat ggc aag gac tac 672 Glu Ser ProAsp Asn Ala Leu Gln Phe Arg Leu Tyr Gly Lys Asp Tyr 210 215 220 aat gtctac aca cat agc ttc ttg tgc tat ggg aag gat cag gca ctc 720 Asn Val TyrThr His Ser Phe Leu Cys Tyr Gly Lys Asp Gln Ala Leu 225 230 235 240 tggcag aaa ctg gcc aag gac att cag gtt gca agt aat gaa att ctc 768 Trp GlnLys Leu Ala Lys Asp Ile Gln Val Ala Ser Asn Glu Ile Leu 245 250 255 agggac cca tgc ttt cat cct gga tat aag aag gta gtg aac gta agt 816 Arg AspPro Cys Phe His Pro Gly Tyr Lys Lys Val Val Asn Val Ser 260 265 270 gacctt tac aag acc ccc tgc acc aag aga ttt gag atg act ctt cca 864 Asp LeuTyr Lys Thr Pro Cys Thr Lys Arg Phe Glu Met Thr Leu Pro 275 280 285 ttccag cag ttt gaa atc cag ggt att gga aac tat caa caa tgc cat 912 Phe GlnGln Phe Glu Ile Gln Gly Ile Gly Asn Tyr Gln Gln Cys His 290 295 300 caaagc atc ctg gag ctc ttc aac acc agt tac tgc cct tac tcc cag 960 Gln SerIle Leu Glu Leu Phe Asn Thr Ser Tyr Cys Pro Tyr Ser Gln 305 310 315 320tgt gcc ttc aat ggg att ttc ttg cca cca ctc cag ggg gat ttt ggg 1008 CysAla Phe Asn Gly Ile Phe Leu Pro Pro Leu Gln Gly Asp Phe Gly 325 330 335gca ttt tca gct ttt tac ttt gtg atg aag ttt tta aac ttg aca tca 1056 AlaPhe Ser Ala Phe Tyr Phe Val Met Lys Phe Leu Asn Leu Thr Ser 340 345 350gag aaa gtc tct cag gaa aag gtg act gag atg atg aaa aag ttc tgt 1104 GluLys Val Ser Gln Glu Lys Val Thr Glu Met Met Lys Lys Phe Cys 355 360 365gct cag cct tgg gag gag ata aaa aca tct tac gct gga gta aag gag 1152 AlaGln Pro Trp Glu Glu Ile Lys Thr Ser Tyr Ala Gly Val Lys Glu 370 375 380aag tac ctg agt gaa tac tgc ttt tct ggt acc tac att ctc tcc ctc 1200 LysTyr Leu Ser Glu Tyr Cys Phe Ser Gly Thr Tyr Ile Leu Ser Leu 385 390 395400 ctt ctg caa ggc tat cat ttc aca gct gat tcc tgg gag cac atc cat 1248Leu Leu Gln Gly Tyr His Phe Thr Ala Asp Ser Trp Glu His Ile His 405 410415 ttc att ggc aag atc cag ggc agc gac gcc ggc tgg act ttg ggc tac 1296Phe Ile Gly Lys Ile Gln Gly Ser Asp Ala Gly Trp Thr Leu Gly Tyr 420 425430 atg ctg aac ctg acc aac atg atc cca gct gag caa cca ttg tcc aca 1344Met Leu Asn Leu Thr Asn Met Ile Pro Ala Glu Gln Pro Leu Ser Thr 435 440445 cct ctc tcc cac tcc acc taa 1365 Pro Leu Ser His Ser Thr 450 6 454PRT Artificial Sequence Description of Artificial Sequence Fusionconstruct of human CD39 6 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr GlnLeu Thr Ser Ser Thr 1 5 10 15 Gln Asn Lys Ala Leu Pro Glu Asn Val LysTyr Gly Ile Val Leu Asp 20 25 30 Ala Gly Ser Ser His Thr Ser Leu Tyr IleTyr Lys Trp Pro Ala Glu 35 40 45 Lys Glu Asn Asp Thr Gly Val Val His GlnVal Glu Glu Cys Arg Val 50 55 60 Lys Gly Pro Gly Ile Ser Lys Phe Val GlnLys Val Asn Glu Ile Gly 65 70 75 80 Ile Tyr Leu Thr Asp Cys Met Glu ArgAla Arg Glu Val Ile Pro Arg 85 90 95 Ser Gln His Gln Glu Thr Pro Val TyrLeu Gly Ala Thr Ala Gly Met 100 105 110 Arg Leu Leu Arg Met Glu Ser GluGlu Leu Ala Asp Arg Val Leu Asp 115 120 125 Val Val Glu Arg Ser Leu SerAsn Tyr Pro Phe Asp Phe Gln Gly Ala 130 135 140 Arg Ile Ile Thr Gly GlnGlu Glu Gly Ala Tyr Gly Trp Ile Thr Ile 145 150 155 160 Asn Tyr Leu LeuGly Lys Phe Ser Gln Lys Thr Arg Trp Phe Ser Ile 165 170 175 Val Pro TyrGlu Thr Asn Asn Gln Glu Thr Phe Gly Ala Leu Asp Leu 180 185 190 Gly GlyAla Ser Thr Gln Val Thr Phe Val Pro Gln Asn Gln Thr Ile 195 200 205 GluSer Pro Asp Asn Ala Leu Gln Phe Arg Leu Tyr Gly Lys Asp Tyr 210 215 220Asn Val Tyr Thr His Ser Phe Leu Cys Tyr Gly Lys Asp Gln Ala Leu 225 230235 240 Trp Gln Lys Leu Ala Lys Asp Ile Gln Val Ala Ser Asn Glu Ile Leu245 250 255 Arg Asp Pro Cys Phe His Pro Gly Tyr Lys Lys Val Val Asn ValSer 260 265 270 Asp Leu Tyr Lys Thr Pro Cys Thr Lys Arg Phe Glu Met ThrLeu Pro 275 280 285 Phe Gln Gln Phe Glu Ile Gln Gly Ile Gly Asn Tyr GlnGln Cys His 290 295 300 Gln Ser Ile Leu Glu Leu Phe Asn Thr Ser Tyr CysPro Tyr Ser Gln 305 310 315 320 Cys Ala Phe Asn Gly Ile Phe Leu Pro ProLeu Gln Gly Asp Phe Gly 325 330 335 Ala Phe Ser Ala Phe Tyr Phe Val MetLys Phe Leu Asn Leu Thr Ser 340 345 350 Glu Lys Val Ser Gln Glu Lys ValThr Glu Met Met Lys Lys Phe Cys 355 360 365 Ala Gln Pro Trp Glu Glu IleLys Thr Ser Tyr Ala Gly Val Lys Glu 370 375 380 Lys Tyr Leu Ser Glu TyrCys Phe Ser Gly Thr Tyr Ile Leu Ser Leu 385 390 395 400 Leu Leu Gln GlyTyr His Phe Thr Ala Asp Ser Trp Glu His Ile His 405 410 415 Phe Ile GlyLys Ile Gln Gly Ser Asp Ala Gly Trp Thr Leu Gly Tyr 420 425 430 Met LeuAsn Leu Thr Asn Met Ile Pro Ala Glu Gln Pro Leu Ser Thr 435 440 445 ProLeu Ser His Ser Thr 450 7 1437 DNA Artificial Sequence Description ofArtificial Sequence Fusion construct of human CD39 7 atg gcc ctg tgg atcgac agg atg caa ctc ctg tct tgc att gca cta 48 Met Ala Leu Trp Ile AspArg Met Gln Leu Leu Ser Cys Ile Ala Leu 1 5 10 15 agt ctt gca ctt gtcaca aac agt gca cct act tca agt tct aca aag 96 Ser Leu Ala Leu Val ThrAsn Ser Ala Pro Thr Ser Ser Ser Thr Lys 20 25 30 aaa aca cag cta act agttca acc cag aac aaa gca ttg cca gaa aac 144 Lys Thr Gln Leu Thr Ser SerThr Gln Asn Lys Ala Leu Pro Glu Asn 35 40 45 gtt aag tat ggg att gtg ctggat gcg ggt tct tct cac aca agt tta 192 Val Lys Tyr Gly Ile Val Leu AspAla Gly Ser Ser His Thr Ser Leu 50 55 60 tac atc tat aag tgg cca gca gaaaag gag aat gac aca ggc gtg gtg 240 Tyr Ile Tyr Lys Trp Pro Ala Glu LysGlu Asn Asp Thr Gly Val Val 65 70 75 80 cat caa gta gaa gaa tgc agg gttaaa ggt cct gga atc tca aaa ttt 288 His Gln Val Glu Glu Cys Arg Val LysGly Pro Gly Ile Ser Lys Phe 85 90 95 gtt cag aaa gta aat gaa ata ggc atttac ctg act gat tgc atg gaa 336 Val Gln Lys Val Asn Glu Ile Gly Ile TyrLeu Thr Asp Cys Met Glu 100 105 110 aga gct agg gaa gtg att cca agg tcccag cac caa gag aca ccc gtt 384 Arg Ala Arg Glu Val Ile Pro Arg Ser GlnHis Gln Glu Thr Pro Val 115 120 125 tac ctg gga gcc acg gca ggc atg cggttg ctc agg atg gaa agt gaa 432 Tyr Leu Gly Ala Thr Ala Gly Met Arg LeuLeu Arg Met Glu Ser Glu 130 135 140 gag ttg gca gac agg gtt ctg gat gtggtg gag agg agc ctc agc aac 480 Glu Leu Ala Asp Arg Val Leu Asp Val ValGlu Arg Ser Leu Ser Asn 145 150 155 160 tac ccc ttt gac ttc cag ggt gccagg atc att act ggc caa gag gaa 528 Tyr Pro Phe Asp Phe Gln Gly Ala ArgIle Ile Thr Gly Gln Glu Glu 165 170 175 ggt gcc tat ggc tgg att act atcaac tat ctg ctg ggc aaa ttc agt 576 Gly Ala Tyr Gly Trp Ile Thr Ile AsnTyr Leu Leu Gly Lys Phe Ser 180 185 190 cag aaa aca agg tgg ttc agc atagtc cca tat gaa acc aat aat cag 624 Gln Lys Thr Arg Trp Phe Ser Ile ValPro Tyr Glu Thr Asn Asn Gln 195 200 205 gaa acc ttt gga gct ttg gac cttggg gga gcc tct aca caa gtc act 672 Glu Thr Phe Gly Ala Leu Asp Leu GlyGly Ala Ser Thr Gln Val Thr 210 215 220 ttt gta ccc caa aac cag act atcgag tcc cca gat aat gct ctg caa 720 Phe Val Pro Gln Asn Gln Thr Ile GluSer Pro Asp Asn Ala Leu Gln 225 230 235 240 ttt cgc ctc tat ggc aag gactac aat gtc tac aca cat agc ttc ttg 768 Phe Arg Leu Tyr Gly Lys Asp TyrAsn Val Tyr Thr His Ser Phe Leu 245 250 255 tgc tat ggg aag gat cag gcactc tgg cag aaa ctg gcc aag gac att 816 Cys Tyr Gly Lys Asp Gln Ala LeuTrp Gln Lys Leu Ala Lys Asp Ile 260 265 270 cag gtt gca agt aat gaa attctc agg gac cca tgc ttt cat cct gga 864 Gln Val Ala Ser Asn Glu Ile LeuArg Asp Pro Cys Phe His Pro Gly 275 280 285 tat aag aag gta gtg aac gtaagt gac ctt tac aag acc ccc tgc acc 912 Tyr Lys Lys Val Val Asn Val SerAsp Leu Tyr Lys Thr Pro Cys Thr 290 295 300 aag aga ttt gag atg act cttcca ttc cag cag ttt gaa atc cag ggt 960 Lys Arg Phe Glu Met Thr Leu ProPhe Gln Gln Phe Glu Ile Gln Gly 305 310 315 320 att gga aac tat caa caatgc cat caa agc atc ctg gag ctc ttc aac 1008 Ile Gly Asn Tyr Gln Gln CysHis Gln Ser Ile Leu Glu Leu Phe Asn 325 330 335 acc agt tac tgc cct tactcc cag tgt gcc ttc aat ggg att ttc ttg 1056 Thr Ser Tyr Cys Pro Tyr SerGln Cys Ala Phe Asn Gly Ile Phe Leu 340 345 350 cca cca ctc cag ggg gatttt ggg gca ttt tca gct ttt tac ttt gtg 1104 Pro Pro Leu Gln Gly Asp PheGly Ala Phe Ser Ala Phe Tyr Phe Val 355 360 365 atg aag ttt tta aac ttgaca tca gag aaa gtc tct cag gaa aag gtg 1152 Met Lys Phe Leu Asn Leu ThrSer Glu Lys Val Ser Gln Glu Lys Val 370 375 380 act gag atg atg aaa aagttc tgt gct cag cct tgg gag gag ata aaa 1200 Thr Glu Met Met Lys Lys PheCys Ala Gln Pro Trp Glu Glu Ile Lys 385 390 395 400 aca tct tac gct ggagta aag gag aag tac ctg agt gaa tac tgc ttt 1248 Thr Ser Tyr Ala Gly ValLys Glu Lys Tyr Leu Ser Glu Tyr Cys Phe 405 410 415 tct ggt acc tac attctc tcc ctc ctt ctg caa ggc tat cat ttc aca 1296 Ser Gly Thr Tyr Ile LeuSer Leu Leu Leu Gln Gly Tyr His Phe Thr 420 425 430 gct gat tcc tgg gagcac atc cat ttc att ggc aag atc cag ggc agc 1344 Ala Asp Ser Trp Glu HisIle His Phe Ile Gly Lys Ile Gln Gly Ser 435 440 445 gac gcc ggc tgg actttg ggc tac atg ctg aac ctg acc aac atg atc 1392 Asp Ala Gly Trp Thr LeuGly Tyr Met Leu Asn Leu Thr Asn Met Ile 450 455 460 cca gct gag caa ccattg tcc aca cct ctc tcc cac tcc acc taa 1437 Pro Ala Glu Gln Pro Leu SerThr Pro Leu Ser His Ser Thr 465 470 475 8 478 PRT Artificial SequenceDescription of Artificial Sequence Fusion construct of human CD39 8 MetAla Leu Trp Ile Asp Arg Met Gln Leu Leu Ser Cys Ile Ala Leu 1 5 10 15Ser Leu Ala Leu Val Thr Asn Ser Ala Pro Thr Ser Ser Ser Thr Lys 20 25 30Lys Thr Gln Leu Thr Ser Ser Thr Gln Asn Lys Ala Leu Pro Glu Asn 35 40 45Val Lys Tyr Gly Ile Val Leu Asp Ala Gly Ser Ser His Thr Ser Leu 50 55 60Tyr Ile Tyr Lys Trp Pro Ala Glu Lys Glu Asn Asp Thr Gly Val Val 65 70 7580 His Gln Val Glu Glu Cys Arg Val Lys Gly Pro Gly Ile Ser Lys Phe 85 9095 Val Gln Lys Val Asn Glu Ile Gly Ile Tyr Leu Thr Asp Cys Met Glu 100105 110 Arg Ala Arg Glu Val Ile Pro Arg Ser Gln His Gln Glu Thr Pro Val115 120 125 Tyr Leu Gly Ala Thr Ala Gly Met Arg Leu Leu Arg Met Glu SerGlu 130 135 140 Glu Leu Ala Asp Arg Val Leu Asp Val Val Glu Arg Ser LeuSer Asn 145 150 155 160 Tyr Pro Phe Asp Phe Gln Gly Ala Arg Ile Ile ThrGly Gln Glu Glu 165 170 175 Gly Ala Tyr Gly Trp Ile Thr Ile Asn Tyr LeuLeu Gly Lys Phe Ser 180 185 190 Gln Lys Thr Arg Trp Phe Ser Ile Val ProTyr Glu Thr Asn Asn Gln 195 200 205 Glu Thr Phe Gly Ala Leu Asp Leu GlyGly Ala Ser Thr Gln Val Thr 210 215 220 Phe Val Pro Gln Asn Gln Thr IleGlu Ser Pro Asp Asn Ala Leu Gln 225 230 235 240 Phe Arg Leu Tyr Gly LysAsp Tyr Asn Val Tyr Thr His Ser Phe Leu 245 250 255 Cys Tyr Gly Lys AspGln Ala Leu Trp Gln Lys Leu Ala Lys Asp Ile 260 265 270 Gln Val Ala SerAsn Glu Ile Leu Arg Asp Pro Cys Phe His Pro Gly 275 280 285 Tyr Lys LysVal Val Asn Val Ser Asp Leu Tyr Lys Thr Pro Cys Thr 290 295 300 Lys ArgPhe Glu Met Thr Leu Pro Phe Gln Gln Phe Glu Ile Gln Gly 305 310 315 320Ile Gly Asn Tyr Gln Gln Cys His Gln Ser Ile Leu Glu Leu Phe Asn 325 330335 Thr Ser Tyr Cys Pro Tyr Ser Gln Cys Ala Phe Asn Gly Ile Phe Leu 340345 350 Pro Pro Leu Gln Gly Asp Phe Gly Ala Phe Ser Ala Phe Tyr Phe Val355 360 365 Met Lys Phe Leu Asn Leu Thr Ser Glu Lys Val Ser Gln Glu LysVal 370 375 380 Thr Glu Met Met Lys Lys Phe Cys Ala Gln Pro Trp Glu GluIle Lys 385 390 395 400 Thr Ser Tyr Ala Gly Val Lys Glu Lys Tyr Leu SerGlu Tyr Cys Phe 405 410 415 Ser Gly Thr Tyr Ile Leu Ser Leu Leu Leu GlnGly Tyr His Phe Thr 420 425 430 Ala Asp Ser Trp Glu His Ile His Phe IleGly Lys Ile Gln Gly Ser 435 440 445 Asp Ala Gly Trp Thr Leu Gly Tyr MetLeu Asn Leu Thr Asn Met Ile 450 455 460 Pro Ala Glu Gln Pro Leu Ser ThrPro Leu Ser His Ser Thr 465 470 475 9 24 PRT Artificial SequenceDescription of Artificial Sequence Synthetic signal sequence 9 Met AlaLeu Trp Ile Asp Arg Met Gln Leu Leu Ser Cys Ile Ala Leu 1 5 10 15 SerLeu Ala Leu Val Thr Asn Ser 20 10 8 PRT Artificial Sequence Descriptionof Artificial Sequence Synthetic peptide 10 Asp Tyr Lys Asp Asp Asp AspLys 1 5 11 43 PRT Artificial Sequence Description of Artificial SequenceFusion construct of human CD39 11 Met Ala Leu Trp Ile Asp Arg Met GlnLeu Leu Ser Cys Ile Ala Leu 1 5 10 15 Ser Leu Ala Leu Val Thr Asn SerAla Pro Thr Ser Ser Ser Thr Lys 20 25 30 Lys Thr Gln Leu Thr Ser Ser ThrGln Asn Lys 35 40 12 29 PRT Artificial Sequence Description ofArtificial Sequence Fusion construct of human CD39 12 Met Ala Leu TrpIle Asp Arg Met Gln Leu Leu Ser Cys Ile Ala Leu 1 5 10 15 Ser Leu AlaLeu Val Thr Asn Ser Ala Thr Gln Asn Lys 20 25 13 31 PRT ArtificialSequence Description of Artificial Sequence Fusion construct of humanCD39 13 Met Ala Leu Trp Ile Asp Arg Met Gln Leu Leu Ser Cys Ile Ala Leu1 5 10 15 Ser Leu Ala Leu Val Thr Asn Ser Ala Ser Ser Thr Gln Asn Lys 2025 30 14 87 DNA Artificial Sequence Description of Artificial SequenceSynthetic oligonucleotide 14 ccggctggac tttgggctac atgctgaacc tgaccaacatgatcccagct gagcaaccat 60 tgtccacacc tctctcccac gagcccc 87 15 87 DNAArtificial Sequence Description of Artificial Sequence Syntheticoligonucleotide 15 gatcggggct cgtgggagag aggtgtggac aatggttgctcagctgggat catgttggtc 60 aggttcagca tgtagcccaa agtccag 87 16 740 DNAHomo sapiens CDS (42)..(737) 16 cggtaccgct agcgtcgaca ggcctaggatatcgatacgt a gag ccc aga tct tgt 56 Glu Pro Arg Ser Cys 1 5 gac aaa actcac aca tgc cca ccg tgc cca gca cct gaa gcc gag ggc 104 Asp Lys Thr HisThr Cys Pro Pro Cys Pro Ala Pro Glu Ala Glu Gly 10 15 20 gcg ccg tca gtcttc ctc ttc ccc cca aaa ccc aag gac acc ctc atg 152 Ala Pro Ser Val PheLeu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 25 30 35 atc tcc cgg acc cctgag gtc aca tgc gtg gtg gtg gac gtg agc cac 200 Ile Ser Arg Thr Pro GluVal Thr Cys Val Val Val Asp Val Ser His 40 45 50 gaa gac cct gag gtc aagttc aac tgg tac gtg gac ggc gtg gag gtg 248 Glu Asp Pro Glu Val Lys PheAsn Trp Tyr Val Asp Gly Val Glu Val 55 60 65 cat aat gcc aag aca aag ccgcgg gag gag cag tac aac agc acg tac 296 His Asn Ala Lys Thr Lys Pro ArgGlu Glu Gln Tyr Asn Ser Thr Tyr 70 75 80 85 cgg gtg gtc agc gtc ctc accgtc ctg cac cag gac tgg ctg aat ggc 344 Arg Val Val Ser Val Leu Thr ValLeu His Gln Asp Trp Leu Asn Gly 90 95 100 aag gac tac aag tgc aag gtctcc aac aaa gcc ctc cca gcc ccc atg 392 Lys Asp Tyr Lys Cys Lys Val SerAsn Lys Ala Leu Pro Ala Pro Met 105 110 115 cag aaa acc atc tcc aaa gccaaa ggg cag ccc cga gaa cca cag gtg 440 Gln Lys Thr Ile Ser Lys Ala LysGly Gln Pro Arg Glu Pro Gln Val 120 125 130 tac acc ctg ccc cca tcc cgggat gag ctg acc aag aac cag gtc agc 488 Tyr Thr Leu Pro Pro Ser Arg AspGlu Leu Thr Lys Asn Gln Val Ser 135 140 145 ctg acc tgc ctg gtc aaa ggcttc tat ccc agg cac atc gcc gtg gag 536 Leu Thr Cys Leu Val Lys Gly PheTyr Pro Arg His Ile Ala Val Glu 150 155 160 165 tgg gag agc aat ggg cagccg gag aac aac tac aag acc acg cct ccc 584 Trp Glu Ser Asn Gly Gln ProGlu Asn Asn Tyr Lys Thr Thr Pro Pro 170 175 180 gtg ctg gac tcc gac ggctcc ttc ttc ctc tac agc aag ctc acc gtg 632 Val Leu Asp Ser Asp Gly SerPhe Phe Leu Tyr Ser Lys Leu Thr Val 185 190 195 gac aag agc agg tgg cagcag ggg aac gtc ttc tca tgc tcc gtg atg 680 Asp Lys Ser Arg Trp Gln GlnGly Asn Val Phe Ser Cys Ser Val Met 200 205 210 cat gag gct ctg cac aaccac tac acg cag aag agc ctc tcc ctg tct 728 His Glu Ala Leu His Asn HisTyr Thr Gln Lys Ser Leu Ser Leu Ser 215 220 225 ccg ggt aaa tga 740 ProGly Lys 230 17 232 PRT Homo sapiens 17 Glu Pro Arg Ser Cys Asp Lys ThrHis Thr Cys Pro Pro Cys Pro Ala 1 5 10 15 Pro Glu Ala Glu Gly Ala ProSer Val Phe Leu Phe Pro Pro Lys Pro 20 25 30 Lys Asp Thr Leu Met Ile SerArg Thr Pro Glu Val Thr Cys Val Val 35 40 45 Val Asp Val Ser His Glu AspPro Glu Val Lys Phe Asn Trp Tyr Val 50 55 60 Asp Gly Val Glu Val His AsnAla Lys Thr Lys Pro Arg Glu Glu Gln 65 70 75 80 Tyr Asn Ser Thr Tyr ArgVal Val Ser Val Leu Thr Val Leu His Gln 85 90 95 Asp Trp Leu Asn Gly LysAsp Tyr Lys Cys Lys Val Ser Asn Lys Ala 100 105 110 Leu Pro Ala Pro MetGln Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 115 120 125 Arg Glu Pro GlnVal Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr 130 135 140 Lys Asn GlnVal Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Arg 145 150 155 160 HisIle Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 165 170 175Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 180 185190 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 195200 205 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys210 215 220 Ser Leu Ser Leu Ser Pro Gly Lys 225 230 18 18 DNA ArtificialSequence Description of Artificial Sequence Synthetic oligonucleotide 18ctttccatcc tgagcaac 18 19 36 DNA Artificial Sequence Description ofArtificial Sequence Synthetic oligonucleotide 19 aaaaaactag tcagaacaaagctttgccag aaaacg 36 20 24 PRT Mus sp. 20 Met Phe His Val Ser Phe ArgTyr Ile Phe Gly Ile Pro Pro Leu Ile 1 5 10 15 Leu Val Leu Leu Pro ValThr Ser 20 21 46 DNA Artificial Sequence Description of ArtificialSequence Synthetic oligonucleotide 21 ctagttctgg agactacaaa gatgacgatgacaaaaccca gaacaa 46 22 46 DNA Artificial Sequence Description ofArtificial Sequence Synthetic oligonucleotide 22 agctttgttc tgggttttgtcatcgtcatc tttgtagtct ccagaa 46 23 89 DNA Artificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 23ccggctggac tttgggctac atgctgaacc tgaccaacat gatcccagct gagcaaccat 60tgtccacacc tctctcccac tccacctaa 89 24 89 DNA Artificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 24ggccttaggt ggagtgggag agaggtgtgg acaatggttg ctcagctggg atcatgttgg 60tcaggttcag catgtagccc aaagtccag 89 25 1464 DNA Artificial Sequence CDS(1)..(1461) Description of Artificial Sequence Fusion construct of humanCD39 25 atg gcc ctg tgg atc gac agg atg caa ctc ctg tct tgc att gca cta48 Met Ala Leu Trp Ile Asp Arg Met Gln Leu Leu Ser Cys Ile Ala Leu 1 510 15 agt ctt gca ctt gtc aca aac agt gca cct act tca agt tct aca aag 96Ser Leu Ala Leu Val Thr Asn Ser Ala Pro Thr Ser Ser Ser Thr Lys 20 25 30aaa aca cag cta act agt tca gga gac tac aaa gat gac gat gac aaa 144 LysThr Gln Leu Thr Ser Ser Gly Asp Tyr Lys Asp Asp Asp Asp Lys 35 40 45 acccag aac aaa gca ttg cca gaa aac gtt aag tat ggg att gtg ctg 192 Thr GlnAsn Lys Ala Leu Pro Glu Asn Val Lys Tyr Gly Ile Val Leu 50 55 60 gat gcgggt tct tct cac aca agt tta tac atc tat aag tgg cca gca 240 Asp Ala GlySer Ser His Thr Ser Leu Tyr Ile Tyr Lys Trp Pro Ala 65 70 75 80 gaa aaggag aat gac aca ggc gtg gtg cat caa gta gaa gaa tgc agg 288 Glu Lys GluAsn Asp Thr Gly Val Val His Gln Val Glu Glu Cys Arg 85 90 95 gtt aaa ggtcct gga atc tca aaa ttt gtt cag aaa gta aat gaa ata 336 Val Lys Gly ProGly Ile Ser Lys Phe Val Gln Lys Val Asn Glu Ile 100 105 110 ggc att tacctg act gat tgc atg gaa aga gct agg gaa gtg att cca 384 Gly Ile Tyr LeuThr Asp Cys Met Glu Arg Ala Arg Glu Val Ile Pro 115 120 125 agg tcc cagcac caa gag aca ccc gtt tac ctg gga gcc acg gca ggc 432 Arg Ser Gln HisGln Glu Thr Pro Val Tyr Leu Gly Ala Thr Ala Gly 130 135 140 atg cgg ttgctc agg atg gaa agt gaa gag ttg gca gac agg gtt ctg 480 Met Arg Leu LeuArg Met Glu Ser Glu Glu Leu Ala Asp Arg Val Leu 145 150 155 160 gat gtggtg gag agg agc ctc agc aac tac ccc ttt gac ttc cag ggt 528 Asp Val ValGlu Arg Ser Leu Ser Asn Tyr Pro Phe Asp Phe Gln Gly 165 170 175 gcc aggatc att act ggc caa gag gaa ggt gcc tat ggc tgg att act 576 Ala Arg IleIle Thr Gly Gln Glu Glu Gly Ala Tyr Gly Trp Ile Thr 180 185 190 atc aactat ctg ctg ggc aaa ttc agt cag aaa aca agg tgg ttc agc 624 Ile Asn TyrLeu Leu Gly Lys Phe Ser Gln Lys Thr Arg Trp Phe Ser 195 200 205 ata gtccca tat gaa acc aat aat cag gaa acc ttt gga gct ttg gac 672 Ile Val ProTyr Glu Thr Asn Asn Gln Glu Thr Phe Gly Ala Leu Asp 210 215 220 ctt ggggga gcc tct aca caa gtc act ttt gta ccc caa aac cag act 720 Leu Gly GlyAla Ser Thr Gln Val Thr Phe Val Pro Gln Asn Gln Thr 225 230 235 240 atcgag tcc cca gat aat gct ctg caa ttt cgc ctc tat ggc aag gac 768 Ile GluSer Pro Asp Asn Ala Leu Gln Phe Arg Leu Tyr Gly Lys Asp 245 250 255 tacaat gtc tac aca cat agc ttc ttg tgc tat ggg aag gat cag gca 816 Tyr AsnVal Tyr Thr His Ser Phe Leu Cys Tyr Gly Lys Asp Gln Ala 260 265 270 ctctgg cag aaa ctg gcc aag gac att cag gtt gca agt aat gaa att 864 Leu TrpGln Lys Leu Ala Lys Asp Ile Gln Val Ala Ser Asn Glu Ile 275 280 285 ctcagg gac cca tgc ttt cat cct gga tat aag aag gta gtg aac gta 912 Leu ArgAsp Pro Cys Phe His Pro Gly Tyr Lys Lys Val Val Asn Val 290 295 300 agtgac ctt tac aag acc ccc tgc acc aag aga ttt gag atg act ctt 960 Ser AspLeu Tyr Lys Thr Pro Cys Thr Lys Arg Phe Glu Met Thr Leu 305 310 315 320cca ttc cag cag ttt gaa atc cag ggt att gga aac tat caa caa tgc 1008 ProPhe Gln Gln Phe Glu Ile Gln Gly Ile Gly Asn Tyr Gln Gln Cys 325 330 335cat caa agc atc ctg gag ctc ttc aac acc agt tac tgc cct tac tcc 1056 HisGln Ser Ile Leu Glu Leu Phe Asn Thr Ser Tyr Cys Pro Tyr Ser 340 345 350cag tgt gcc ttc aat ggg att ttc ttg cca cca ctc cag ggg gat ttt 1104 GlnCys Ala Phe Asn Gly Ile Phe Leu Pro Pro Leu Gln Gly Asp Phe 355 360 365ggg gca ttt tca gct ttt tac ttt gtg atg aag ttt tta aac ttg aca 1152 GlyAla Phe Ser Ala Phe Tyr Phe Val Met Lys Phe Leu Asn Leu Thr 370 375 380tca gag aaa gtc tct cag gaa aag gtg act gag atg atg aaa aag ttc 1200 SerGlu Lys Val Ser Gln Glu Lys Val Thr Glu Met Met Lys Lys Phe 385 390 395400 tgt gct cag cct tgg gag gag ata aaa aca tct tac gct gga gta aag 1248Cys Ala Gln Pro Trp Glu Glu Ile Lys Thr Ser Tyr Ala Gly Val Lys 405 410415 gag aag tac ctg agt gaa tac tgc ttt tct ggt acc tac att ctc tcc 1296Glu Lys Tyr Leu Ser Glu Tyr Cys Phe Ser Gly Thr Tyr Ile Leu Ser 420 425430 ctc ctt ctg caa ggc tat cat ttc aca gct gat tcc tgg gag cac atc 1344Leu Leu Leu Gln Gly Tyr His Phe Thr Ala Asp Ser Trp Glu His Ile 435 440445 cat ttc att ggc aag atc cag ggc agc gac gcc ggc tgg act ttg ggc 1392His Phe Ile Gly Lys Ile Gln Gly Ser Asp Ala Gly Trp Thr Leu Gly 450 455460 tac atg ctg aac ctg acc aac atg atc cca gct gag caa cca ttg tcc 1440Tyr Met Leu Asn Leu Thr Asn Met Ile Pro Ala Glu Gln Pro Leu Ser 465 470475 480 aca cct ctc tcc cac tcc acc taa 1464 Thr Pro Leu Ser His Ser Thr485 26 487 PRT Artificial Sequence Description of Artificial SequenceFusion construct of human CD39 26 Met Ala Leu Trp Ile Asp Arg Met GlnLeu Leu Ser Cys Ile Ala Leu 1 5 10 15 Ser Leu Ala Leu Val Thr Asn SerAla Pro Thr Ser Ser Ser Thr Lys 20 25 30 Lys Thr Gln Leu Thr Ser Ser GlyAsp Tyr Lys Asp Asp Asp Asp Lys 35 40 45 Thr Gln Asn Lys Ala Leu Pro GluAsn Val Lys Tyr Gly Ile Val Leu 50 55 60 Asp Ala Gly Ser Ser His Thr SerLeu Tyr Ile Tyr Lys Trp Pro Ala 65 70 75 80 Glu Lys Glu Asn Asp Thr GlyVal Val His Gln Val Glu Glu Cys Arg 85 90 95 Val Lys Gly Pro Gly Ile SerLys Phe Val Gln Lys Val Asn Glu Ile 100 105 110 Gly Ile Tyr Leu Thr AspCys Met Glu Arg Ala Arg Glu Val Ile Pro 115 120 125 Arg Ser Gln His GlnGlu Thr Pro Val Tyr Leu Gly Ala Thr Ala Gly 130 135 140 Met Arg Leu LeuArg Met Glu Ser Glu Glu Leu Ala Asp Arg Val Leu 145 150 155 160 Asp ValVal Glu Arg Ser Leu Ser Asn Tyr Pro Phe Asp Phe Gln Gly 165 170 175 AlaArg Ile Ile Thr Gly Gln Glu Glu Gly Ala Tyr Gly Trp Ile Thr 180 185 190Ile Asn Tyr Leu Leu Gly Lys Phe Ser Gln Lys Thr Arg Trp Phe Ser 195 200205 Ile Val Pro Tyr Glu Thr Asn Asn Gln Glu Thr Phe Gly Ala Leu Asp 210215 220 Leu Gly Gly Ala Ser Thr Gln Val Thr Phe Val Pro Gln Asn Gln Thr225 230 235 240 Ile Glu Ser Pro Asp Asn Ala Leu Gln Phe Arg Leu Tyr GlyLys Asp 245 250 255 Tyr Asn Val Tyr Thr His Ser Phe Leu Cys Tyr Gly LysAsp Gln Ala 260 265 270 Leu Trp Gln Lys Leu Ala Lys Asp Ile Gln Val AlaSer Asn Glu Ile 275 280 285 Leu Arg Asp Pro Cys Phe His Pro Gly Tyr LysLys Val Val Asn Val 290 295 300 Ser Asp Leu Tyr Lys Thr Pro Cys Thr LysArg Phe Glu Met Thr Leu 305 310 315 320 Pro Phe Gln Gln Phe Glu Ile GlnGly Ile Gly Asn Tyr Gln Gln Cys 325 330 335 His Gln Ser Ile Leu Glu LeuPhe Asn Thr Ser Tyr Cys Pro Tyr Ser 340 345 350 Gln Cys Ala Phe Asn GlyIle Phe Leu Pro Pro Leu Gln Gly Asp Phe 355 360 365 Gly Ala Phe Ser AlaPhe Tyr Phe Val Met Lys Phe Leu Asn Leu Thr 370 375 380 Ser Glu Lys ValSer Gln Glu Lys Val Thr Glu Met Met Lys Lys Phe 385 390 395 400 Cys AlaGln Pro Trp Glu Glu Ile Lys Thr Ser Tyr Ala Gly Val Lys 405 410 415 GluLys Tyr Leu Ser Glu Tyr Cys Phe Ser Gly Thr Tyr Ile Leu Ser 420 425 430Leu Leu Leu Gln Gly Tyr His Phe Thr Ala Asp Ser Trp Glu His Ile 435 440445 His Phe Ile Gly Lys Ile Gln Gly Ser Asp Ala Gly Trp Thr Leu Gly 450455 460 Tyr Met Leu Asn Leu Thr Asn Met Ile Pro Ala Glu Gln Pro Leu Ser465 470 475 480 Thr Pro Leu Ser His Ser Thr 485 27 464 PRT ArtificialSequence Description of Artificial Sequence Fusion construct of humanCD39 27 Met Ala Leu Trp Ile Asp Arg Met Gln Leu Leu Ser Cys Ile Ala Leu1 5 10 15 Ser Leu Ala Leu Val Thr Asn Ser Ala Thr Gln Asn Lys Ala LeuPro 20 25 30 Glu Asn Val Lys Tyr Gly Ile Val Leu Asp Ala Gly Ser Ser HisThr 35 40 45 Ser Leu Tyr Ile Tyr Lys Trp Pro Ala Glu Lys Glu Asn Asp ThrGly 50 55 60 Val Val His Gln Val Glu Glu Cys Arg Val Lys Gly Pro Gly IleSer 65 70 75 80 Lys Phe Val Gln Lys Val Asn Glu Ile Gly Ile Tyr Leu ThrAsp Cys 85 90 95 Met Glu Arg Ala Arg Glu Val Ile Pro Arg Ser Gln His GlnGlu Thr 100 105 110 Pro Val Tyr Leu Gly Ala Thr Ala Gly Met Arg Leu LeuArg Met Glu 115 120 125 Ser Glu Glu Leu Ala Asp Arg Val Leu Asp Val ValGlu Arg Ser Leu 130 135 140 Ser Asn Tyr Pro Phe Asp Phe Gln Gly Ala ArgIle Ile Thr Gly Gln 145 150 155 160 Glu Glu Gly Ala Tyr Gly Trp Ile ThrIle Asn Tyr Leu Leu Gly Lys 165 170 175 Phe Ser Gln Lys Thr Arg Trp PheSer Ile Val Pro Tyr Glu Thr Asn 180 185 190 Asn Gln Glu Thr Phe Gly AlaLeu Asp Leu Gly Gly Ala Ser Thr Gln 195 200 205 Val Thr Phe Val Pro GlnAsn Gln Thr Ile Glu Ser Pro Asp Asn Ala 210 215 220 Leu Gln Phe Arg LeuTyr Gly Lys Asp Tyr Asn Val Tyr Thr His Ser 225 230 235 240 Phe Leu CysTyr Gly Lys Asp Gln Ala Leu Trp Gln Lys Leu Ala Lys 245 250 255 Asp IleGln Val Ala Ser Asn Glu Ile Leu Arg Asp Pro Cys Phe His 260 265 270 ProGly Tyr Lys Lys Val Val Asn Val Ser Asp Leu Tyr Lys Thr Pro 275 280 285Cys Thr Lys Arg Phe Glu Met Thr Leu Pro Phe Gln Gln Phe Glu Ile 290 295300 Gln Gly Ile Gly Asn Tyr Gln Gln Cys His Gln Ser Ile Leu Glu Leu 305310 315 320 Phe Asn Thr Ser Tyr Cys Pro Tyr Ser Gln Cys Ala Phe Asn GlyIle 325 330 335 Phe Leu Pro Pro Leu Gln Gly Asp Phe Gly Ala Phe Ser AlaPhe Tyr 340 345 350 Phe Val Met Lys Phe Leu Asn Leu Thr Ser Glu Lys ValSer Gln Glu 355 360 365 Lys Val Thr Glu Met Met Lys Lys Phe Cys Ala GlnPro Trp Glu Glu 370 375 380 Ile Lys Thr Ser Tyr Ala Gly Val Lys Glu LysTyr Leu Ser Glu Tyr 385 390 395 400 Cys Phe Ser Gly Thr Tyr Ile Leu SerLeu Leu Leu Gln Gly Tyr His 405 410 415 Phe Thr Ala Asp Ser Trp Glu HisIle His Phe Ile Gly Lys Ile Gln 420 425 430 Gly Ser Asp Ala Gly Trp ThrLeu Gly Tyr Met Leu Asn Leu Thr Asn 435 440 445 Met Ile Pro Ala Glu GlnPro Leu Ser Thr Pro Leu Ser His Ser Thr 450 455 460 28 474 PRTArtificial Sequence Description of Artificial Sequence Fusion constructof human CD39 28 Met Ala Leu Trp Ile Asp Arg Met Gln Leu Leu Ser Cys IleAla Leu 1 5 10 15 Ser Leu Ala Leu Val Thr Asn Ser Ala Ser Thr Lys LysThr Gln Leu 20 25 30 Thr Ser Ser Thr Gln Asn Lys Ala Leu Pro Glu Asn ValLys Tyr Gly 35 40 45 Ile Val Leu Asp Ala Gly Ser Ser His Thr Ser Leu TyrIle Tyr Lys 50 55 60 Trp Pro Ala Glu Lys Glu Asn Asp Thr Gly Val Val HisGln Val Glu 65 70 75 80 Glu Cys Arg Val Lys Gly Pro Gly Ile Ser Lys PheVal Gln Lys Val 85 90 95 Asn Glu Ile Gly Ile Tyr Leu Thr Asp Cys Met GluArg Ala Arg Glu 100 105 110 Val Ile Pro Arg Ser Gln His Gln Glu Thr ProVal Tyr Leu Gly Ala 115 120 125 Thr Ala Gly Met Arg Leu Leu Arg Met GluSer Glu Glu Leu Ala Asp 130 135 140 Arg Val Leu Asp Val Val Glu Arg SerLeu Ser Asn Tyr Pro Phe Asp 145 150 155 160 Phe Gln Gly Ala Arg Ile IleThr Gly Gln Glu Glu Gly Ala Tyr Gly 165 170 175 Trp Ile Thr Ile Asn TyrLeu Leu Gly Lys Phe Ser Gln Lys Thr Arg 180 185 190 Trp Phe Ser Ile ValPro Tyr Glu Thr Asn Asn Gln Glu Thr Phe Gly 195 200 205 Ala Leu Asp LeuGly Gly Ala Ser Thr Gln Val Thr Phe Val Pro Gln 210 215 220 Asn Gln ThrIle Glu Ser Pro Asp Asn Ala Leu Gln Phe Arg Leu Tyr 225 230 235 240 GlyLys Asp Tyr Asn Val Tyr Thr His Ser Phe Leu Cys Tyr Gly Lys 245 250 255Asp Gln Ala Leu Trp Gln Lys Leu Ala Lys Asp Ile Gln Val Ala Ser 260 265270 Asn Glu Ile Leu Arg Asp Pro Cys Phe His Pro Gly Tyr Lys Lys Val 275280 285 Val Asn Val Ser Asp Leu Tyr Lys Thr Pro Cys Thr Lys Arg Phe Glu290 295 300 Met Thr Leu Pro Phe Gln Gln Phe Glu Ile Gln Gly Ile Gly AsnTyr 305 310 315 320 Gln Gln Cys His Gln Ser Ile Leu Glu Leu Phe Asn ThrSer Tyr Cys 325 330 335 Pro Tyr Ser Gln Cys Ala Phe Asn Gly Ile Phe LeuPro Pro Leu Gln 340 345 350 Gly Asp Phe Gly Ala Phe Ser Ala Phe Tyr PheVal Met Lys Phe Leu 355 360 365 Asn Leu Thr Ser Glu Lys Val Ser Gln GluLys Val Thr Glu Met Met 370 375 380 Lys Lys Phe Cys Ala Gln Pro Trp GluGlu Ile Lys Thr Ser Tyr Ala 385 390 395 400 Gly Val Lys Glu Lys Tyr LeuSer Glu Tyr Cys Phe Ser Gly Thr Tyr 405 410 415 Ile Leu Ser Leu Leu LeuGln Gly Tyr His Phe Thr Ala Asp Ser Trp 420 425 430 Glu His Ile His PheIle Gly Lys Ile Gln Gly Ser Asp Ala Gly Trp 435 440 445 Thr Leu Gly TyrMet Leu Asn Leu Thr Asn Met Ile Pro Ala Glu Gln 450 455 460 Pro Leu SerThr Pro Leu Ser His Ser Thr 465 470 29 473 PRT Artificial SequenceDescription of Artificial Sequence Fusion construct of human CD39 29 MetAla Leu Trp Ile Asp Arg Met Gln Leu Leu Ser Cys Ile Ala Leu 1 5 10 15Ser Leu Ala Leu Val Thr Asn Ser Ser Thr Lys Lys Thr Gln Leu Thr 20 25 30Ser Ser Thr Gln Asn Lys Ala Leu Pro Glu Asn Val Lys Tyr Gly Ile 35 40 45Val Leu Asp Ala Gly Ser Ser His Thr Ser Leu Tyr Ile Tyr Lys Trp 50 55 60Pro Ala Glu Lys Glu Asn Asp Thr Gly Val Val His Gln Val Glu Glu 65 70 7580 Cys Arg Val Lys Gly Pro Gly Ile Ser Lys Phe Val Gln Lys Val Asn 85 9095 Glu Ile Gly Ile Tyr Leu Thr Asp Cys Met Glu Arg Ala Arg Glu Val 100105 110 Ile Pro Arg Ser Gln His Gln Glu Thr Pro Val Tyr Leu Gly Ala Thr115 120 125 Ala Gly Met Arg Leu Leu Arg Met Glu Ser Glu Glu Leu Ala AspArg 130 135 140 Val Leu Asp Val Val Glu Arg Ser Leu Ser Asn Tyr Pro PheAsp Phe 145 150 155 160 Gln Gly Ala Arg Ile Ile Thr Gly Gln Glu Glu GlyAla Tyr Gly Trp 165 170 175 Ile Thr Ile Asn Tyr Leu Leu Gly Lys Phe SerGln Lys Thr Arg Trp 180 185 190 Phe Ser Ile Val Pro Tyr Glu Thr Asn AsnGln Glu Thr Phe Gly Ala 195 200 205 Leu Asp Leu Gly Gly Ala Ser Thr GlnVal Thr Phe Val Pro Gln Asn 210 215 220 Gln Thr Ile Glu Ser Pro Asp AsnAla Leu Gln Phe Arg Leu Tyr Gly 225 230 235 240 Lys Asp Tyr Asn Val TyrThr His Ser Phe Leu Cys Tyr Gly Lys Asp 245 250 255 Gln Ala Leu Trp GlnLys Leu Ala Lys Asp Ile Gln Val Ala Ser Asn 260 265 270 Glu Ile Leu ArgAsp Pro Cys Phe His Pro Gly Tyr Lys Lys Val Val 275 280 285 Asn Val SerAsp Leu Tyr Lys Thr Pro Cys Thr Lys Arg Phe Glu Met 290 295 300 Thr LeuPro Phe Gln Gln Phe Glu Ile Gln Gly Ile Gly Asn Tyr Gln 305 310 315 320Gln Cys His Gln Ser Ile Leu Glu Leu Phe Asn Thr Ser Tyr Cys Pro 325 330335 Tyr Ser Gln Cys Ala Phe Asn Gly Ile Phe Leu Pro Pro Leu Gln Gly 340345 350 Asp Phe Gly Ala Phe Ser Ala Phe Tyr Phe Val Met Lys Phe Leu Asn355 360 365 Leu Thr Ser Glu Lys Val Ser Gln Glu Lys Val Thr Glu Met MetLys 370 375 380 Lys Phe Cys Ala Gln Pro Trp Glu Glu Ile Lys Thr Ser TyrAla Gly 385 390 395 400 Val Lys Glu Lys Tyr Leu Ser Glu Tyr Cys Phe SerGly Thr Tyr Ile 405 410 415 Leu Ser Leu Leu Leu Gln Gly Tyr His Phe ThrAla Asp Ser Trp Glu 420 425 430 His Ile His Phe Ile Gly Lys Ile Gln GlySer Asp Ala Gly Trp Thr 435 440 445 Leu Gly Tyr Met Leu Asn Leu Thr AsnMet Ile Pro Ala Glu Gln Pro 450 455 460 Leu Ser Thr Pro Leu Ser His SerThr 465 470 30 463 PRT Artificial Sequence Description of ArtificialSequence Fusion construct of human CD39 30 Met Glu Thr Asp Thr Leu LeuLeu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Ala ProThr Ser Thr Gln Asn Lys Ala Leu Pro Glu 20 25 30 Asn Val Lys Tyr Gly IleVal Leu Asp Ala Gly Ser Ser His Thr Ser 35 40 45 Leu Tyr Ile Tyr Lys TrpPro Ala Glu Lys Glu Asn Asp Thr Gly Val 50 55 60 Val His Gln Val Glu GluCys Arg Val Lys Gly Pro Gly Ile Ser Lys 65 70 75 80 Phe Val Gln Lys ValAsn Glu Ile Gly Ile Tyr Leu Thr Asp Cys Met 85 90 95 Glu Arg Ala Arg GluVal Ile Pro Arg Ser Gln His Gln Glu Thr Pro 100 105 110 Val Tyr Leu GlyAla Thr Ala Gly Met Arg Leu Leu Arg Met Glu Ser 115 120 125 Glu Glu LeuAla Asp Arg Val Leu Asp Val Val Glu Arg Ser Leu Ser 130 135 140 Asn TyrPro Phe Asp Phe Gln Gly Ala Arg Ile Ile Thr Gly Gln Glu 145 150 155 160Glu Gly Ala Tyr Gly Trp Ile Thr Ile Asn Tyr Leu Leu Gly Lys Phe 165 170175 Ser Gln Lys Thr Arg Trp Phe Ser Ile Val Pro Tyr Glu Thr Asn Asn 180185 190 Gln Glu Thr Phe Gly Ala Leu Asp Leu Gly Gly Ala Ser Thr Gln Val195 200 205 Thr Phe Val Pro Gln Asn Gln Thr Ile Glu Ser Pro Asp Asn AlaLeu 210 215 220 Gln Phe Arg Leu Tyr Gly Lys Asp Tyr Asn Val Tyr Thr HisSer Phe 225 230 235 240 Leu Cys Tyr Gly Lys Asp Gln Ala Leu Trp Gln LysLeu Ala Lys Asp 245 250 255 Ile Gln Val Ala Ser Asn Glu Ile Leu Arg AspPro Cys Phe His Pro 260 265 270 Gly Tyr Lys Lys Val Val Asn Val Ser AspLeu Tyr Lys Thr Pro Cys 275 280 285 Thr Lys Arg Phe Glu Met Thr Leu ProPhe Gln Gln Phe Glu Ile Gln 290 295 300 Gly Ile Gly Asn Tyr Gln Gln CysHis Gln Ser Ile Leu Glu Leu Phe 305 310 315 320 Asn Thr Ser Tyr Cys ProTyr Ser Gln Cys Ala Phe Asn Gly Ile Phe 325 330 335 Leu Pro Pro Leu GlnGly Asp Phe Gly Ala Phe Ser Ala Phe Tyr Phe 340 345 350 Val Met Lys PheLeu Asn Leu Thr Ser Glu Lys Val Ser Gln Glu Lys 355 360 365 Val Thr GluMet Met Lys Lys Phe Cys Ala Gln Pro Trp Glu Glu Ile 370 375 380 Lys ThrSer Tyr Ala Gly Val Lys Glu Lys Tyr Leu Ser Glu Tyr Cys 385 390 395 400Phe Ser Gly Thr Tyr Ile Leu Ser Leu Leu Leu Gln Gly Tyr His Phe 405 410415 Thr Ala Asp Ser Trp Glu His Ile His Phe Ile Gly Lys Ile Gln Gly 420425 430 Ser Asp Ala Gly Trp Thr Leu Gly Tyr Met Leu Asn Leu Thr Asn Met435 440 445 Ile Pro Ala Glu Gln Pro Leu Ser Thr Pro Leu Ser His Ser Thr450 455 460 31 58 PRT Homo sapiens 31 Met Ala Thr Ser Trp Gly Thr ValPhe Phe Met Leu Val Val Ser Cys 1 5 10 15 Val Cys Ser Ala Val Ser HisArg Asn Gln Gln Thr Trp Phe Glu Gly 20 25 30 Ile Phe Leu Ser Ser Met CysPro Ile Asn Val Ser Ala Ser Thr Leu 35 40 45 Tyr Gly Ile Met Phe Asp AlaGly Ser Thr 50 55

We claim:
 1. A polypeptide having the structure X-Y wherein X isselected from the group consisting of an Ala residue and heterologouspeptides capable of adopting a stable secondary structure and Y is asoluble CD39 polypeptide selected from the group consisting of: (a)polypeptides having an amino acid sequence as set forth in FIG. 1 (SEQID NO:2) wherein the amino terminus is selected from the groupconsisting of amino acids 36-44, and the carboxy terminus is selectedfrom the group consisting of amino acids 471-478; (b) fragments of thepolypeptides of (a) wherein said fragments have apyrase activity; and(c) variants of the polypeptides of (a) or (b), wherein said variantshave apyrase activity.
 2. The polypeptide of claim 1 wherein Y is asoluble CD39 polypeptide selected from the group consisting of: (a)polypeptides having a sequence consisting of amino acids 38-476 or39-476 of SEQ ID NO:2; (b) variant polypeptides that are at least 70%identical in amino acid sequence to amino acids 36 to 478 of SEQ ID NO:2or to a fragment thereof, wherein said variant polypeptides have apyraseactivity; (c) variant polypeptides that are at least 80% identical inamino acid sequence to amino acids 36 to 478 of SEQ ID NO:2 or to afragment thereof, wherein said variant polypeptides have apyraseactivity; (d) variant polypeptides that are at least 90% identical inamino acid sequence to amino acids 36 to 478 of SEQ ID NO:2 or to afragment thereof, wherein said variant polypeptides have apyraseactivity; (e) variant polypeptides that are at least 95% identical inamino acid sequence to amino acids 36 to 478 of SEQ ID NO:2 or to afragment thereof, wherein said variant polypeptides have apyraseactivity; (f) variant polypeptides that are at least 98% identical inamino acid sequence to amino acids 36 to 478 of SEQ ID NO:2 or to afragment thereof, wherein said variant polypeptides have apyraseactivity; and (g) variant polypeptides that are at least 99% identicalin amino acid sequence to amino acids 36 to 478 of SEQ ID NO:2 or to afragment thereof, wherein said variant polypeptides have apyraseactivity.
 3. The polypeptide of claim 1 wherein X is a peptide fragmentfrom the amino terminal portion of mature IL-2, CD39-L2, CD39-L3, orCD39-L4.
 4. A polypeptide having the structure A-B-Y wherein A is 0-20amino acids from the amino terminal portion of mature IL-2, B is alinker of 0-15 amino acids, and Y is a soluble CD39 polypeptide selectedfrom the group consisting of: (a) polypeptides having an amino acidsequence as set forth in FIG. 1 (SEQ ID NO:2) wherein the amino terminusis selected from the group consisting of amino acids 36-44, and thecarboxy terminus is selected from the group consisting of amino acids471-478; (b) fragments of the polypeptides of (a) wherein said fragmentshave apyrase activity; and (c) variants of the polypeptides of (a) or(b), wherein said variants have apyrase activity.
 5. A soluble CD39polypeptide comprising a sequence selected from the group consisting of:(a) SEQ ID NO: 6, amino acids 25-464 of SEQ ID NO:27, amino acids 25-474of SEQ ID NO:28, amino acids 27-473 of SEQ ID NO:29, amino acids 21-476of SEQ ID NO:3, amino acids 21-476 of SEQ ID NO:4, or amino acids 21-463of SEQ ID NO:30; and (b) fusion polypeptides comprising the polypeptidesof (a), wherein said fusion polypeptides have apyrase activity.
 6. Thesoluble CD39 polypeptide of claim 5 having an amino acid sequenceselected from the group consisting of SEQ ID NO: 6, amino acids 25-464of SEQ ID NO:27, amino acids 25-474 of SEQ ID NO:28, amino acids 27-473of SEQ ID NO:29, amino acids 21-476 of SEQ ID NO:3, amino acids 21-476of SEQ ID NO:4, and amino acids 21-463 of SEQ ID NO:30.
 7. The solubleCD39 polypeptide of claim 6 having the sequence of amino acids 21-463 ofSEQ ID NO:30.
 8. An isolated nucleic acid encoding a polypeptide ofclaim
 1. 9. The nucleic acid of claim 8 wherein said nucleic acid isDNA.
 10. The DNA of claim 9 having a sequence selected from the groupconsisting of: (a) SEQ ID NO:5; and (b) DNA sequences which, due todegeneracy of the genetic code, encode the polypeptide encoded by SEQ IDNO:5.
 11. The DNA of claim 9 wherein said DNA further encodes a leaderpeptide operably linked to the N-terminus of the polypeptide, whereinthe leader peptide facilitates the extracellular secretion of thepolypeptide.
 12. The DNA of claim 11 wherein the leader peptidecomprises all or part of a leader from IL-2, proinsulin, human growthhormone (huGH), L7, or Igkappa.
 13. The DNA of claim 12 wherein theleader peptide comprises the sequence SEQ ID NO:9.
 14. The DNA of claim11 having a sequence selected from the group consisting of (a) SEQ IDNO:7; and (b) DNA sequences which, due to degeneracy of the geneticcode, encode the polypeptide encoded by SEQ ID NO:7.
 15. A vectorcomprising the nucleic acid of claim
 8. 16. The vector of claim 15wherein said vector is a eukaryotic expression vector.
 17. A recombinantcell comprising the nucleic acid of claim
 8. 18. The cell of claim 17wherein said cell is a prokaryotic cell.
 19. The cell of claim 17wherein said cell is a eukaryotic cell.
 20. The cell of claim 19 whereinsaid cell is a COS cell or a CHO cell.
 21. The cell of claim 20 whereinsaid cell is a CHO cell that has been adapted to grow in suspension andin the absence of serum.
 22. A process for preparing a soluble CD39polypeptide comprising culturing a recombinant cell according to claim17 under conditions that permit expression of the CD39 polypeptide andrecovering the CD39 polypeptide from the culture.
 23. The process ofclaim 22 wherein the recombinant cell is a eukaryotic cell.
 24. Theprocess of claim 22 wherein the recombinant cell is a CHO cell that hasbeen adapted to grow in suspension and in the absence of serum.
 25. Apolypeptide produced according to the process of claim
 22. 26. Apolypeptide produced according to the process of claim
 24. 27. Acomposition comprising a pharmaceutically acceptable carrier and apolypeptide according to claim
 1. 28. A composition comprising apharmaceutically acceptable carrier and a polypeptide according to claim5.
 29. A composition comprising a pharmaceutically acceptable carrierand a polypeptide according to claim
 25. 30. A method of inhibitingangiogenesis in a mammal in need of such treatment comprisingadministering a therapeutic amount of a soluble CD39 polypeptide.